Exendin analog formulations

ABSTRACT

Novel formulations containing exendins, exendin agonists and/or exendin analogs are provided.

RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 11/083,730 filed Mar. 18, 2005, which is acontinuation of U.S. patent application Ser. No. 09/889,331, filed Dec.18, 2001, now U.S. Pat. No. 6,872,700, which claims the benefit ofInternational Patent Application Serial No. PCT/US00/00942, filed Jan.14, 2000, now abandoned, which claims the benefit of U.S. ProvisionalApplication 60/116,380, filed Jan. 14, 1999, now abandoned, U.S.Provisional Application 60/132,017, filed Apr. 30, 1999, now abandoned,and U.S. Provisional Application 60/175,365, filed Jan. 10, 2000, nowabandoned, the contents of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present application relates to novel formulations containingexendins, exendin agonists and exendin analogs.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT DISCS

The sequence listing in the present application is being submitted ontwo compact discs labeled “Sequence Listing Copy 1” and “SequenceListing Copy 2”; each containing a file of 153 KB in size named“249-167-CIP_Sequence_Listing.txt” created on Sep. 23, 2005, thecontents of which are hereby incorporated by reference.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art to the presently claimedinvention, or relevant, nor that any of the publications specifically orimplicitly referenced are prior art.

The exendins are peptides that are found in the salivary secretions ofthe Gila monster and the Mexican Beaded Lizard, reptiles that areendogenous to Arizona and Northern Mexico. Exendin-3 [SEQ. ID. NO. 1:His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser-NH₂] is present in the salivary secretions of Helodermahorridum (Mexican Beaded Lizard), and exendin-4 [SEQ. ID. NO. 2: His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro ProSer-NH₂] is present in the salivary secretions of Heloderma suspectum(Gila monster)(Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng,J., et al., J. Biol. Chem., 267:7402-05, 1992). The amino acid sequenceof exendin-3 is shown in FIG. 1. The amino acid sequence of exendin-4 isshown in FIG. 2. Exendin-4 was first thought to be a (potentially toxic)component of the venom. It now appears that exendin-4 is devoid oftoxicity, and that it instead is made in salivary glands in the Gilamonster.

The exendins have some sequence similarity to several members of theglucagon-like peptide family, with the highest homology, 53%, being toGLP-1[7-36]NH₂ [SEQ. ID. NO. 3] (Goke, et al., J. Biol. Chem.,268:19650-55, 1993). GLP-1[7-36]NH₂, also sometimes referred to asproglucagon[78-107] or simply “GLP-1” as used most often herein, has aninsulinotropic effect, stimulating insulin secretion from pancreaticbeta-cells; GLP-1 has also been reported to inhibit glucagon secretionfrom pancreatic alpha-cells (Ørsov, et al., Diabetes, 42:658-61, 1993;D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). GLP-1 has beenreported to inhibit gastric emptying (Willms B, et al., J ClinEndocrinol Metab 81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci38 (4): 665-73, 1993), and gastric acid secretion (Schjoldager B T, etal., Dig Dis Sci 34 (5): 703-8, 1989; O'Halloran D J, et al., JEndocrinol 126 (1): 169-73, 1990; Wettergren A, et al., Dig Dis Sci 38(4): 665-73, 1993)). GLP-1[7-37], which has an additional glycineresidue at its carboxy terminus, is reported to stimulate insulinsecretion in humans (Ørskov, et al., Diabetes, 42:658-61, 1993). Atransmembrane G-protein adenylate-cyclase-coupled receptor said to beresponsible at least in part for the insulinotropic effect of GLP-1 hasreportedly been cloned from a beta-cell line (Thorens, Proc. Natl. Acad.Sci. USA 89:8641-45, 1992). GLP-1 has been the focus of significantinvestigation in recent years due to its reported action on theamplification of stimulated insulin production (Byrne M M, Goke B.Lessons from human studies with glucagon-like peptide-1: Potential ofthe gut hormone for clinical use. In: Fehmann H C, Goke B.Insulinotropic Gut Hormone Glucagon-Like Peptide 1. Basel, Switzerland:Karger, 1997:219-33).

Other reports relate to the inhibition of gastric emptying (WettergrenA, et al., Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastricand pancreatic functions in man, Dig. Dis. Sci. 1993 April;38(4):665-73), inhibition of glucagon secretion (Creutzfeldt W O C, etal., Glucagonostatic actions and reduction of fasting hyperglycemia byexogenous glucagon-like peptide I(7-36) amide in type I diabeticpatients, Diabetes Care 1996; 19(6):580-6), and a purported role inappetite control (Turton M D, et al., A role for glucagon-like peptide-1in the central regulation of feeding, Nature 1996 January;379(6560):69-72).

GLP-1 has also been reported to restore islet glucose sensitivity inaging rats, restoring their glucose tolerance to that of younger rats(Egan J M, et al., Glucagon-like peptide-1 restores acute-phase insulinrelease to aged rats, Diabetologia 1997 June; 40(Suppl 1):A130).However, the short duration of biological action of GLP-1 in vivo is onefeature of the peptide that has hampered its development as atherapeutic agent. Various methods have been tried to prolong thehalf-life of GLP-1 or GLP-1(7-37), including attempts to alter theiramino acid sequence and to deliver them using certain formulations (see,e.g., European Patent Application, entitled “Prolonged Delivery ofPeptides,” by Darley, et al., publication number 0 619 322 A2, regardingthe inclusion of polyethylene glycol in formulations containing GLP-1(7-37)).

Pharmacological studies have led to reports that exendin-4 can act atGLP-1 receptors on certain insulin-secreting cells, at dispersed acinarcells from guinea pig pancreas, and at parietal cells from stomach; thepeptide is also reported to stimulate somatostatin release and inhibitgastrin release in isolated stomachs (Goke, et al., J. Biol. Chem.268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91, 1994;Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and exendin-4were reportedly found to stimulate cAMP production in, and amylaserelease from, pancreatic acinar cells (Malhotra, R., et al., RegulatoryPeptides, 41:149-56, 1992; Raufman, et al., J. Biol. Chem. 267:21432-37,1992; Singh, et al., Regul. Pept. 53:47-59, 1994). Additionally,exendin-4 has a significantly longer duration of action than GLP-1. Forexample, in one experiment, glucose lowering by exendin-4 in diabeticmice was reported to persist for several hours, and, depending on dose,for up to 24 hours (Eng J. Prolonged effect of exendin-4 onhyperglycemia of db/db mice, Diabetes 1996 May; 45(Suppl 2):152A(abstract 554)). Based on their insulinotropic activities, the use ofexendin-3 and exendin-4 for the treatment of diabetes mellitus and theprevention of hyperglycemia has been proposed (Eng, U.S. Pat. No.5,424,286).

The results of an investigation of whether exendins are the specieshomolog of mammalian GLP-1 was reported by Chen and Drucker who clonedthe exendin gene from the Gila monster (J. Biol. Chem. 272(7):4108-15(1997)). The observation that the Gila monster also has separate genesfor proglucagons (from which GLP-1 is processed), that are more similarto mammalian proglucagon than exendin, indicates that exendins are notmerely species homologs of GLP-1.

To date, agents that serve to delay gastric emptying have generallyfound a place in medicine as diagnostic aids in gastrointestinalradiological examinations. For example, glucagon is a polypeptidehormone that is produced by the alpha cells of the pancreatic islets ofLangerhans. It is a hyperglycemic agent that mobilizes glucose byactivating hepatic glycogenolysis. It can to a lesser extent stimulatethe secretion of pancreatic insulin. Glucagon is used in the treatmentof insulin-induced hypoglycemia, for example, when administration ofglucose intravenously is not possible. However, as glucagon reduces themotility of the gastro-intestinal tract it is also used as a diagnosticaid in gastrointestinal radiological examinations. Glucagon has alsobeen used in several studies to treat various painful gastrointestinaldisorders associated with spasm. Daniel, et al. (Br. Med. J., 3:720,1974) reported quicker symptomatic relief of acute diverticulitis inpatients treated with glucagon compared with those who had been treatedwith analgesics or antispasmodics. A review by Glauser, et al. (J. Am.Coll. Emergency Physns, 8:228, 1979) described relief of acuteesophageal food obstruction following glucagon therapy. In anotherstudy, glucagon significantly relieved pain and tenderness in 21patients with biliary tract disease compared with 22 patients treatedwith placebo (M. J. Stower, et al., Br. J. Surg., 69:591-2, 1982).

Methods for regulating gastrointestinal motility using amylin agonistsare described in commonly owned International Application No.PCT/US94/10225, published Mar. 16, 1995.

Methods for regulating gastrointestinal motility using exendin agonistsare described in commonly owned U.S. patent application Ser. No.08/908,867, filed Aug. 8, 1997 entitled “Methods for RegulatingGastrointestinal Motility,” which application is a continuation-in-partof U.S. patent application Ser. No. 08/694,954, filed Aug. 8, 1996.

Methods for reducing food intake using exendin agonists are described incommonly owned U.S. patent application Ser. No. 09/003,869, filed Jan.7, 1998, entitled “Use of Exendin and Agonists Thereof for the Reductionof Food Intake,” which claims the benefit of U.S. ProvisionalApplication Nos. 60/034,905 filed Jan. 7, 1997, 60/055,404 filed Aug. 7,1997, 60/065,442 filed Nov. 14, 1997 and 60/066,029 filed Nov. 14, 1997.

Novel exendin agonist compounds are described in commonly owned PCTApplication Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997.

Other novel exendin agonists are described in commonly owned PCTApplication Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/065,442 filed Nov. 14, 1997.

Still other novel exendin agonists are described in commonly owned PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/066,029 filed Nov. 14, 1997.

Other recent advances in exendin related technology are described inU.S. Provisional Patent Application Ser. No. 60/075,122, filed Feb. 13,1998, entitled “Inotropic and Diuretic Effects of Exendin and GLP-1” andin U.S. Provisional Patent Application Ser. No. 60/116,380, filed Jan.14, 1998, entitled “Novel Exendin Agonist Formulations and Methods ofAdministration Thereof”.

Polyethylene glycol (PEG) modification of therapeutic peptides andproteins may yield both advantages and disadvantages. While PEGmodification may lead to improved circulation time, reduced antigenicityand immunogenicity, improved solubility, resistance to proteolysis,improved bioavailability, reduced toxicity, improved stability, andeasier formulation of peptides (See, Francis et al., InternationalJournal of Hematology, 68:1-18, 1998) problems with PEGylation in mostcases is substantial reduction in bioactivity. Id. In addition, mostmethods involve use of linkers that have several types of adverseeffects including immunogenicity, instability, toxicity, and reactivity.Id.

Glucagonoma (tumor of glucagon-secreting cells) produces, in addition toglucose intolerance, a skin condition, necrolytic migratory erythema.This is a raised scaly red rash, sometimes blistering and eventuallycrusting, localized to the face, abdomen, extremities and perineum. Itcan also be associated with inflamation of the tongue and mouth, anddiseased nails and thinning of the hair. The condition is reported torespond to octreotide, a glucagonostatic hormone analog. The compoundsdescribed herein are also useful as glucagonastatic agents and thus inthe treatment of this disease, which was was first described in 1966(Kaplan, L. M. Endocrine Tumors of the Gastrointestinal Tract andPancreas. Ch 262, p 1392: In Harrison's Principles of Internal Medicine,12th Edition. McGraw-Hill Inc, New York, 1991). The compounds describedherein that are useful for lowering glucagon levels and/or suppressingglucagon secretion include exendin, exendin agonists, and modifiedexendins and exendin agonists and related formulations, and dosageformulations.

The contents of the above-identified articles, patents, and patentapplications, and all other documents mentioned or cited herein, arehereby incorporated by reference in their entirety. Applicants reservethe right to physically incorporate into this application any and allmaterials and information from any such articles, patents, patentapplications, or other documents mentioned or cited herein.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides novel exendinagonist compound formulations and dosages thereof exhibitingadvantageous properties that include effects in slowing gastric emptyingand lowering plasma glucose levels. Thus, this aspect of the inventionincludes formulations of exendins and exendin agonists that comprise anexendin or exendin agonist mixed together with a buffer (preferably anacetate buffer), an iso-osmolality modifier (preferably mannitol), andoptionally containing a preservative (preferably m-cresol), saidformulation having a pH of between about 3.0 and about 7.0 (preferablybetween about 4.0 and about 5.0).

Additional formulations within the scope of the invention include aparenteral liquid dosage form, a lyophilized unit-dosage form, alyophilized multi-use dosage form, and modifications of these dosageforms that are useful in the oral, nasal, buccal, sublingual,intra-tracheal, and pulmonary delivery of exendins and exendin agonists.

Thus, the invention includes parenteral liquid dosage forms thatcomprise approximately 0.005 to about 0.4%, more specifically from about0.005 to about 0.02%, or from about 0.005 to about 0.05% (w/v),respectively of the active ingredient in an aqueous system along withapproximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate orglutamate or similar buffer either alone or in combination to obtain apH of the final composition of approximately 3.0 to 7.0, morespecifically from about pH 4.0 to about 6.0, or from about 4.0 to 5.0,as well as either approximately 1.0 to 10% (w/v) of a carbohydrate orpolyhydric alcohol iso-osmolality modifier (preferably mannitol) or upto about 0.9% saline or a combination of both leading to an isotonic oran iso-osmolar solution in an aqueous continuous phase. Approximately0.005 to 1.0% (w/v) of an anti-microbial preservative selected from thegroup consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl andbutyl parabens and phenol is also present if the formulation is packagedin a multi-use container. A sufficient amount of water for injection isadded to obtain the desired concentration of solution. Sodium chloride,as well as other excipients, may also be present, if desired. Suchexcipients, however, must maintain the overall stability of the activeingredient. Useful polyhydric alcohols include such compounds assorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). Thepolyhydric alcohols and the carbohydrates will also be effective instabilizing protein against denaturation caused by elevated temperatureand by freeze-thaw or freeze-drying processes. Suitable carbohydratesinclude galactose, arabinose, lactose or any other carbohydrate whichdoes not have an adverse affect on a diabetic patient, if intended forthat use, i.e., the carbohydrate is not metabolized to form largeconcentrations of glucose in the blood. Preferably, the peptides of thepresent invention are admixed with a polyhydric alcohol such assorbitol, mannitol, inositol, glycerol, xylitol, andpolypropylene/ethylene glycol copolymer, as well as various polyethyleneglycols (PEG) of molecular weight 200, 400, 1450, 3350, 4000, 6000, and8000). Mannitol is the preferred polyhydric alcohol.

The lyophilized unit-dose formulations of the present invention are alsostable, but need not be isotonic and/or iso-osmolar. They include activeingredient(s), a bulking agent to facilitate cake formation (which mayalso act as a tonicifer and/or iso-osmolality modifier uponreconstitution to either facilitate stability of the active ingredientand/or lessen the pain on injection), and may also include a surfactantthat benefits the properties of the cake and/or facilitatesreconstitution. The lyophilized unit-dose formulations of the presentinvention include approximately 0.005 to about 0.4%, more specificallyfrom about 0.005 to about 0.02%, or 0.005 to 0.05% (w/v) of the activeingredient. It may not be necessary to include a buffer in theformulation and/or to reconstitute the lyophile with a buffer if theintention is to consume the contents of the container within thestability period established for the reconstituted active ingredient. Ifa buffer is used, it may be included in the lyophile or in thereconstitution solvent. Therefore, the formulation and/or thereconstitution solvent may contain individually or collectivelyapproximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate orglutamate buffer either alone or in combination to obtain a pH of thefinal composition of approximately 3.0 to 7.0, more specifically fromabout pH 4.0 to about 6.0, or from about 4.0 to 5.0. The bulking agentmay consist of either approximately 1.0 to 10% (w/v) of a carbohydrateor polyhydric alcohol iso-osmolality modifier (as described above) or upto 0.9% saline or a combination of both leading to a isotonic oriso-osmolar solution in the reconstituted aqueous phase. A surfactant,preferably about 0.1 to about 1.0% (w/v) of polysorbate 80 or othernon-ionic detergent, may be included. As noted above, sodium chloride,as well as other excipients, may also be present in the lyophilizedunit-dosage formulation, if desired. The liquid formulation of theinvention prior to lyophilization will be substantially isotonic and/oriso-osmolar either before lyophilization or to enable formation ofisotonic and/or iso-osmolar solutions after reconstitution.

The invention also includes lyophilized and liquid multi-doseformulations. As with the parenteral liquid and lyophilized unit-dosageformulations described above, the lyophilized multi-unit-dosage formshould contain a bulking agent to facilitate cake formation. Apreservative is included to facilitate multiple use by the patient.These dosage forms include approximately 0.005 to about 0.4%, morespecifically from about 0.005 to about 0.02%, or from about 0.005 to0.05% (w/v), respectively of the active ingredient. If a buffer is used,it may be included in the lyophile or in the reconstitution solvent, andthe formulation and/or the reconstitution solvent may containindividually or collectively approximately 0.02 to 0.5% (w/v) of anacetate, phosphate, citrate or glutamate buffer either alone or incombination to obtain a pH of the final composition of approximately 3.0to 7.0, more specifically from about pH 4.0 to about 6.0, or from about4.0 to 5.0. The bulking agent may consist of either approximately 1.0 to10% (w/v) of a carbohydrate or a polyhydric alcohol iso-osmolalitymodifier (preferably mannitol) or up to 0.9% saline, or a combination ofboth, leading to an isotonic or iso-osmolar solution in thereconstituted aqueous phase. A surfactant, preferably about 0.1 to about1.0% (w/v) of polysorbate 80 or other non-ionic detergent, may beincluded. Approximately 0.005 to 1.0% (w/v) of an anti-microbialpreservative selected from the group consisting of m-cresol, benzylalcohol, methyl, ethyl, propyl and butyl parabens and phenol (preferablym-cresol) is also present if the formulation is packaged in a multi-usecontainer. Sodium chloride, as well as other excipients, may also bepresent, if desired. The liquid formulation of the invention should besubstantially isotonic and/or iso-osmolar either before lyophilizationor to enable formation of isotonic and/or iso-osmolar solutions afterreconstitution.

The invention further includes solid dosage forms useful for oral,buccal, sublingual, intra-tracheal, nasal, and pulmonary delivery. Theformulations that best support pulmonary and/or intra-tracheal dosageforms may be either preserved or unpreserved liquid formulations and/ordry powder formulations. The preserved or unpreserved liquidformulations will be essentially identical to the formulations describedabove under preserved or unpreserved liquid parenteral formulations. ThepH of the solution should be about 3.0 to 7.0, more specifically fromabout 4.0 to 6.0, or from about 4.0 to 5.0, with a pH greater than orequal to about 5.0 being most preferred to reduce the potential forbronchoconstriction. The dry powder formulations may contain a bulkingagent and/or salts to facilitate particle size formation and appropriateparticle size distribution. A surfactant and/or salts may also benefitthe properties of the particle morphology and/or facilitate tissueuptake of the active ingredient. Dry powder dosage forms can range from1% to 100% (w/w), respectively of the active ingredient. It may not benecessary to include a bulking agent and/or salts to facilitate particlesize formation and/or distribution. The bulking agent and/or salts mayconsist of either approximately 0 to 99% (w/w) of a carbohydrate orpolyhydric alcohol or approximately 0 to 99% salt or a combination ofboth leading to the preferred particle size and distribution. Asurfactant, preferably about 0.1 to about 1.0% (w/w) of polysorbate 80or other non-ionic detergent, may be included. Sodium chloride, as wellas other excipients, may also be present, if desired. Such excipients,however, will maintain the overall stability of the active ingredientand facilitate the proper level of hydration.

Also within the scope of the invention is the formulation comprising upto 50 mg/ml of an exendin or an exendin agonist in 30 mM acetate buffer(pH about 4.5) and mannitol, with or without a preservative.

Further within the scope of the invention are preferred dosages forexendins and exendin agonists when given by injection, and when given byother routes. Thus, formulations for exendin and exendin agonists havingcomparable potency are provided for the administration by injection offrom about 0.1 to about 0.5 μg per kilogram, given one to three timesper day. Typically, for the patient with diabetes who weighs in therange from about 70 kilograms (average for the type 1 diabetic) to about90 kilograms (average for the type 2 diabetic), for example, this willresult in the total administration of about 10 to about 120 μg per dayin single or divided doses. If administered in divided doses, the dosesare preferably administered two or three times per day, and morepreferably, two times per day.

Oral dosages according to the present invention will include from about50 to about 100 times the active ingredient, i.e., from about 500 toabout 12,000 μg per day in single or divided doses, preferably fromabout 500 to about 5,000 μg per day. Pulmonary dosages according to thepresent invention will include from about 10 to about 100 times theactive ingredient, i.e., from about 100 to about 12,000 μg per day insingle or divided doses, preferably about 500 to 1000 μg per day. Nasal,buccal and sublingual dosages according to the present invention willalso include from about 10 to about 100 times the active ingredient,i.e., from about 100 to about 12,000 μg per day in single or divideddoses.

Preferred dosages for nasal administration are from about 10-1000 toabout 1200-12,000 μg per day, for buccal administration from about10-1000 to about 1200-12,000 μg per day, and for sublingualadministration from about 10-1000 to about 1200-8,000 μg per day.Sublingual dosages are preferably smaller than buccal dosages.Administration dosages for exendin agonists having less than or greaterthan the potency of exendin-4 are increased or decreased as appropriatefrom those described above and elsewhere herein.

Also included within the scope of the present invention are methods ofadministration of said novel exendin agonist compound formulations anddosages by delivery means alternative to subcutaneous injection orintravenous infusion, including, for example, by nasal delivery,pulmonary delivery, oral delivery, intra-tracheal delivery, sublingualdelivery, and buccal delivery.

According to another aspect, the present invention provides novelexendin agonist compound formulations and dosages, and methods for theadministration thereof, that are useful in treating diabetes (includingtype 1 and type 2 diabetes), obesity, and other conditions that willbenefit from the administration of a therapy that can slow gastricemptying, lowering plasma glucose levels, and reduce food intake.

Preferred exendins or exendin agonists for use include:

exendin-4 (1-30) [SEQ ID NO 4: His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Gly];

exendin-4 (1-30) amide [SEQ ID NO 5: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn Gly Gly-NH₂];

exendin-4 (1-28) amide [SEQ ID NO 6: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn-NH₂];

¹⁴Leu, ²⁵Phe exendin-4 amide [SEQ ID NO 7: His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu PheLeu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH₂];

¹⁴Leu, ²⁵Phe exendin-4 (1-28) amide [SEQ ID NO 8: His Gly Glu Gly ThrPhe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe IleGlu Phe Leu Lys Asn-NH₂]; and

¹⁴Leu, ²²Ala, ²⁵Phe exendin-4 (1-28) amide [SEQ ID NO 9: His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu AlaIle Glu Phe Leu Lys Asn-NH₂].

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise.

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers iftheir structure allow such stereoisomeric forms. Natural amino acidsinclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited toazetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,N-methylglycine, N-methylisoleucine, N-methylpentylglycine,N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,pentylglycine, pipecolic acid and thioproline. Amino acid analogsinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or modified on their N-terminalamino group or their side-chain groups, as for example, methioninesulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone.

The term “amino acid analog” refers to an amino acid wherein either theC-terminal carboxy group, the N-terminal amino group or side-chainfunctional group has been chemically codified to another functionalgroup. For example, aspartic acid-(beta-methyl ester) is an amino acidanalog of aspartic acid; N-ethylglycine is an amino acid analog ofglycine; or alanine carboxamide is an amino acid analog of alanine.

The term “amino acid residue” refers to radicals having the structure:(1) —C(O)—R—NH—, wherein R typically is —CH(R′)—, wherein R′ is an aminoacid side chain, typically H or a carbon containing substitutent;or

wherein p is 1, 2 or 3 representing the azetidinecarboxylic acid,proline or pipecolic acid residues, respectively.

The term “lower” referred to herein in connection with organic radicalssuch as alkyl groups defines such groups with up to and including about6, preferably up to and including 4 and advantageously one or two carbonatoms. Such groups may be straight chain or branched chain.

“Pharmaceutically acceptable salt” includes salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid. In practice the use of the salt formamounts to use of the base form. The compounds of the present inventionare useful in both free base and salt form, with both forms beingconsidered as being within the scope of the present invention.

In addition, the following abbreviations stand for the following:

“ACN” or “CH₃CN” refers to acetonitrile.

“Boc”, “tBoc” or “Tboc” refers to t-butoxy carbonyl.

“DCC” refers to N,N′-dicyclohexylcarbodiimide.

“Fmoc” refers to fluorenylmethoxycarbonyl.

“HBTU” refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexaflurophosphate.

“HOBt” refers to 1-hydroxybenzotriazole monohydrate.

“homoP” or hPro” refers to homoproline.

“MeAla” or “Nme” refers to N-methylalanine.

“naph” refers to naphthylalanine.

“pG” or pGly” refers to pentylglycine.

“tBuG” refers to tertiary-butylglycine.

“ThioP” or tpro” refers to thioproline.

“3Hyp” refers to 3-hydroxyproline

“4Hyp” refers to 4-hydroxyproline

“NAG” refers to N-alkylglycine

“NAPG” refers to N-alkylpentylglycine

“Norval” refers to norvaline

“Norleu” refers to norleucine

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence for exendin-3 [SEQ. ID. NO. 1].

FIG. 2 depicts the amino acid sequence for exendin-4 [SEQ. ID. NO. 2].

FIG. 3 depicts the amino acid sequences for certain exendin agonistcompounds useful in the present invention [SEQ. ID. NOS. 10 to 40].

FIG. 4 depicts the amino acid sequences for certain compounds of thepresent invention, Compounds 1-174.

FIG. 5 is a graph showing the effect of functional nephrectomy onexendin-4 clearance.

FIG. 6 is a graph showing the terminal decay of exendin-4 plasma levelsin nephrectomized and sham subjects.

FIG. 7 depicts the plasma levels of exendin-4 in rats afterintra-tracheal administration.

FIG. 8 a depicts the plasma exendin-4 concentration after intra-trachealinstillation in db/db mice.

FIG. 8 b depicts the effect of intra-tracheal administration ofexendin-4 on plasma glucose in db/db mice.

FIGS. 9 a and 9 b depict the effect of intra-tracheal administration ofexendin-4 on plasma glucose in ob/ob mice.

FIG. 10 a depicts the plasma exendin-4 concentration afterintra-tracheal instillation into rats.

FIG. 10 b depicts the bioavailability of exendin-4 followingintra-tracheal instillation into rats.

FIG. 11 depicts plasma exendin-4 concentrations in rats exposed toaerosolized exendin-4 (8 ng/ml) for 10 minutes.

FIG. 12 a depicts the effect of ten minutes of exposure to aerosolizedexendin-4 on plasma glucose in db/db mice.

FIG. 12 b depicts the plasma exendin-4 concentration after ten minutesof exposure of db/db mice to aerosolized exendin-4.

FIG. 13 depicts plasma exendin-4 concentrations in rats afterintra-nasal administration of exendin-4.

FIG. 14 depicts the effect of intra-gastric administration of exendin-4on plasma glucose in db/db mice.

FIG. 15 a depicts the plasma exendin-4 concentration after sublingualadministration to db/db mice.

FIG. 15 b depicts the effect of sublingual administration of exendin-4on plasma glucose in db/db mice.

FIG. 15 c depicts the plasma exendin-4 concentration after sublingualadministration to rats.

FIG. 15 d depicts the bioavailability of exendin-4 after sublingualadministration.

FIG. 15 e depicts the Cmax of sublingual exendin-4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to relates to methods of suppressingand/or lowering glucagon in a subject, comprising the administration ofan exendin, an exendin agonist, or a modified exendin or exendin agonisthaving an exendin or exendin agonist peptide linked to one or morepolyethylene glycol polymers or other compound useful to increasemolecular weight. Such methods are useful, for example, in the treatmentof hyperglucagonemia and other conditions in which lower levels ofglucagon or suppresion of glucagon secretion are of benefit. Suchconditions include, but are not limited to, glucagonoma and necrolyticmigratory erythema.

Modified Exendins and Exendin Agonists

The modified exendins and exendin agonists of the present inventioninclude, for example, one or more PEG polymers linked to an exendin orexendin agonist, such as a naturally occuring exendin, a syntheticexendin or an exendin agonist.

Exendin-4

Exendin-4 is a naturally occurring peptide isolated from the salivarysecretions of the Gila monster. Animal testing of exendin-4 has shownthat its ability to lower blood glucose persists for several hours.Exendin-4, a 39-amino acid polypeptide, is synthesized using solid phasesynthesis as described herein, and this synthetic material has beenshown to be identical to that of native exendin-4.

As described herein, the nonclinical pharmacology of exendin-4 has beenstudied. In the brain, exendin-4 binds principally to the area postremaand nucleus tractus solitarius region in the hindbrain and to thesubfornical organ in the forebrain. Exendin-4 binding has been observedin the rat and mouse brain and kidney. The structures to which exendin-4binds in the kidney are unknown.

Various experiments have compared the biologic actions of exendin-4 andGLP-1 and demonstrated a more favorable spectrum of properties forexendin-4. A single subcutaneous dose of exendin-4 lowered plasmaglucose in db/db (diabetic) and ob/ob (diabetic obese) mice by up to40%. In Diabetic Fatty Zucker (ZDF) rats, 5 weeks of treatment withexendin-4 lowered HbA_(1c) (a measure of glycosylated hemoglobin used toevaluate plasma glucose levels) by up to 41%. Insulin sensitivity wasalso improved by 76% following 5 weeks of treatment in obese ZDF rats.In glucose intolerant primates, dose-dependent decreases in plasmaglucose were also observed.

An insulinotropic action of exendin-4 has also been observed in rodents,improving insulin response to glucose by over 100% in non-fasted HarlanSprague Dawley (HSD) rats, and by up to ˜10-fold in non-fasted db/dbmice. Higher pretreatment plasma glucose concentrations were associatedwith greater glucose-lowering effects. Thus the observed glucoselowering effect of exendin-4 appears to be glucose-dependent, andminimal if animals are already euglycemic.

Exendin-4 dose dependently slowed gastric emptying in HSD rats and was˜90-fold more potent than GLP-1 for this action. Exendin-4 has also beenshown to reduce food intake in NIH/Sw (Swiss) mice following peripheraladministration, and was at least 1000 times more potent than GLP-1 forthis action. Exendin-4 reduced plasma glucagon concentrations byapproximately 40% in anesthetized ZDF rats during hyperinsulinemic,hyperglycemic clamp conditions, but did not affect plasma glucagonconcentrations during euglycemic conditions in normal rats. Exendin-4has been shown to dose-dependently reduce body weight in obese ZDF rats,while in lean ZDF rats, the observed decrease in body weight appears tobe transient.

Through effects on lowering glucagon and supressing glucagon secretion,exendins, exendin agonists, and modified exendins or exendin agonistscontaining exendin-4, for example, will be useful in people who wouldbenefit from lowered glucagon, for example, people with glucagonoma andnecrolytic migratory erythema, and people with diabetes whether or notthey retain the ability to secrete insulin. See Example 5.

The toxicology of exendin-4 has been investigated in single-dose studiesin mice, rats and monkeys, repeated-dose (up to 28 consecutive dailydoses) studies in rats and monkeys and in vitro tests for mutagenicityand chromosomal alterations. To date, no deaths have occurred, and therehave been no observed treatment-related changes in hematology, clinicalchemistry, or gross or microscopic tissue changes. Exendin-4 wasdemonstrated to be non-mutagenic, and did not cause chromosomalaberrations at the concentrations tested (up to 5000 μg/mL).

In support of the investigation of the nonclinical pharmacokinetics andmetabolism of exendin-4, a number of immunoassays have been developed. Aradioimmunoassay with limited sensitivity (˜100 pM) was used in initialpharmacokinetic studies. A two-site IRMA assay for exendin-4 wassubsequently validated with a lower limit of quantitation of 15 pM. Thebioavailability of exendin-4, given subcutaneously, was found to beapproximately 50-80% using the radioimmunoassay. This was similar tothat seen following intraperitoneal administration (48-60%). Peak plasmaconcentrations (C_(max)) occurred between 30 and 43 minutes (T_(max)).Both C_(max) and AUC values were monotonically related to dose. Theapparent terminal half-life for exendin-4 given subcutaneously wasapproximately 90-110 minutes. This was significantly longer than the14-41 minutes seen following intravenous dosing. Similar results wereobtained using the IRMA assay. Degradation studies with exendin-4compared to GLP-1 indicate that exendin-4 is relatively resistant todegradation.

Exendin Agonists

The structure activity relationship (SAR) of exendin was investigatedfor structures that may relate to the antidiabetic activity of exendin,for its stability to metabolism, and for improvement of its physicalcharacteristics, especially as it pertains to peptide stability and toamenability to alternative delivery systems, and various exendin agonistpeptide compounds have been invented. Exendin agonists include exendinpeptide analogs in which one or more naturally occurring amino acids areeliminated or replaced with another amino acid(s). Preferred exendinagonists are agonist analogs of exendin-4. Particularly preferredexendin agonists include those described in commonly owned PCTApplication Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997; commonlyowned PCT Application Serial No. PCT/US98/24210, filed Nov. 13, 1998,entitled “Novel Exendin Agonist Compounds,” which claims the benefit ofU.S. Provisional Application No. 60/065,442 filed Nov. 14, 1997; and,commonly owned PCT Application Serial No. PCT/US98/24273, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds,” which claims thebenefit of U.S. Provisional Application No. 60/066,029 filed Nov. 14,1997, all of which are incorporated herein by reference in theirentirety, including any drawings.

Activity as exendin agonists can be indicated, for example, by activityin the assays described below. Effects of exendins or exendin agonistson gastric motility and gastric emptying can be identified, evaluated,or screened for, using the methods described herein, or other art-knownor equivalent methods for determining gastric motility. Negativereceptor assays or screens for exendin agonist compounds or candidateexendin agonist compounds, such as an amylin receptor assay/screen usingan amylin receptor preparation as described in U.S. Pat. No. 5,264,372,issued Nov. 23, 1993, the contents of which are incorporated herein byreference, one or more calcitonin receptor assays/screens using, forexample, T47D and MCF7 breast carcinoma cells, which contain calciumreceptors coupled to the stimulation of adenyl cyclase activity, and/ora CGRP receptor assay/screen using, for example, SK-N-MC cells.

One such method for use in identifying or evaluating the ability of acompound to slow gastric motility, involves: (a) bringing together atest sample and a test system, the test sample containing one or moretest compounds, the test system containing a system for evaluatinggastric motility, the system being characterized in that it exhibits,for example, elevated plasma glucose in response to the introduction tothe system of glucose or a meal; and, (b) determining the presence oramount of a rise in plasma glucose in the system. Positive and/ornegative controls may be used as well.

Also included within the scope of the present invention arepharmaceutically acceptable salts of the compounds of formula (I-VIII)and pharmaceutical compositions including said compounds and saltsthereof.

Formula I

Exendin agonist compounds also include those described in U.S.Provisional Application No. 60/065,442, including compounds of theformula (I) [SEQ ID NO. 41]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁; wherein

-   Xaa₁ is His, Arg or Tyr;-   Xaa₂ is Ser, Gly, Ala or Thr;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe, Tyr or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Asp or Glu;-   Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala or Leu;-   Xaa₂₂ is Ala, Phe, Tyr or naphthylalanine;-   Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;-   Xaa₂₆ is Ala or Leu;-   Xaa₂₇ is Ala or Lys;-   Xaa₂₈ is Ala or Asn;-   Z₁ is —OH,    -   —NH₂    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,        N-alkylpentylglycine or N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala.        Preferred N-alkyl groups for N-alkylglycine,        N-alkylpentylglycine and N-alkylalanine include lower alkyl        groups preferably of 1 to about 6 carbon atoms, more preferably        of 1 to 4 carbon atoms.

Preferred exendin agonist compounds include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycine orMet.

Preferred compounds are those wherein Xaa₂₅ is Trp or Phe.

Preferred compounds are those where Xaa₆ is Phe or naphthylalanine;Xaa₂₂ is Phe or naphthylalanine and Xaa₂₃ is Ile or Val.

Preferred are compounds wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (I) whereinXaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ is Phe ornaphthylalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Phe ornaphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (I) wherein: Xaa₁ is His or Arg; Xaa₂is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₅ is Ala or Thr; Xaa₆ is Ala, Pheor nephthylalaine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ isAsp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala or Ser;Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu orpentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ SerSer-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Pro homoproline,thioproline or N-methylalanine; and Z₂ being —OH or —NH₂; provided thatno more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂,Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅,Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala. Especially preferred compounds includethose set forth in PCT application Serial No. PCT/US98/24210, filed Nov.13, 1998, entitled “Novel Exendin Agonist Compounds” identified thereinas compounds 2-23.

According to an especially preferred aspect, provided are compoundswhere Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds will be less susceptive tooxidative degration, both in vitro and in vivo, as well as duringsynthesis of the compound.

Formula II

Exendin agonist compounds also include those described in U.S.Provisional Application No. 60/066,029, including compounds of theformula (II)[SEQ ID NO. 42]:

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁; wherein

-   Xaa₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu;-   Xaa₂ is Ser, Gly, Ala or Thr;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₄ is Ala, Norval, Val, Norleu or Gly;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe, Tyr or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;-   Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala or Leu;-   Xaa₂₂ is Phe, Tyr or naphthylalanine;-   Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;-   Xaa₂₆ is Ala or Leu;-   Xaa₂₇ is Ala or Lys;-   Xaa₂₈ is Ala or Asn;-   Z₁ is —OH,    -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;    -   wherein    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,        N-alkylpentylglycine or N-alkylalanine;    -   Xaa₃₉ is Ser or Tyr; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆,        Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,        Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈        are Ala; and provided also that, if Xaa₁ is His, Arg or Tyr,        then at least one of Xaa₃, Xaa₄ and Xaa₉ is Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compoundsof formula (II) include those described in application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, identified therein in Examples 1-89 (“Compounds 1-89,”respectively), as well as those corresponding compounds identifiedtherein in Examples 104 and 105.

Preferred such exendin agonist compounds include those wherein Xaa₁ isHis, Ala or Norval. More preferably Xaa₁ is His or Ala. Most preferablyXaa₁ is His.

Preferred are those compounds of formula (II) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (II) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (II) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (II) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (II) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (II) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (II) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (II) include thosedescribed in application Serial No. PCT/US98/24273, filed Nov. 13, 1998,entitled “Novel Exendin Agonist Compounds” as having the amino acidsequence of SEQ. ID. NOS. 5-93 therein.

According to an especially preferred aspect, provided are compounds offormula (II) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula III

Also within the scope of the present invention are narrower genera ofcompounds having peptides of various lengths, for example genera ofcompounds which do not include peptides having a length of 28, 29 or 30amino acid residues, respectively. Additionally, the present inventionincludes narrower genera of compounds described in PCT applicationSerial No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” and having particular amino acid sequences, forexample, compounds of the formula (III) [SEQ. ID. NO. 43]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;

wherein

-   Xaa₁ is His or Arg;-   Xaa₂ is Gly or Ala;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Asp or Glu;-   Xaa₁₀ is Ala, Leu or pentylglycine;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu or pentylglycine;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala or Leu;-   Xaa₂₂ is Phe or naphthylalanine;-   Xaa₂₃ is Ile, Val or tert-butylglycine;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp, or Phe;-   Xaa₂₆ is Ala or Leu;-   Xaa₂₇ is Ala or Lys;-   Xaa₂₈ is Ala or Asn;-   Z₁ is —OH,    -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, thioproline and        N-methylylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and        pharmaceutically acceptable salts thereof.        Formula IV

Additionally, the present invention includes narrower genera of peptidecompounds described in PCT Application Serial No. PCT/US98/24273, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as havingparticular amino acid sequences, for example, compounds of the formula[IV] [SEQ. ID. NO. 44]:

Xaa₁ Xaa₂ Xaa₃ Xaa₅ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁; wherein

-   Xaa₁ is His or Ala;-   Xaa₂ is Gly or Ala;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₄ is Ala or Gly;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Ala, Asp or Glu;-   Xaa₁₀ is Ala, Leu or pentylglycine;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu, Met or pentylglycine;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala or Leu;-   Xaa₂₂ is Phe or naphthylalanine;-   Xaa₂₃ is Ile, Val or tert-butylglycine;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp or Phe;-   Xaa₂₆ is Ala or Leu;-   Xaa₂₇ is Ala or Lys;-   Xaa₂₈ is Ala or Asn;-   Z₁ is —OH,    -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂-   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Ser-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, thioproline, or N-methylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇, and Xaa₂₈ are Ala; and        provided that, if Xaa₁ is His then at least one of Xaa₃, Xaa₄        and Xaa₉ is Ala; and pharmaceutically acceptable salts thereof.

Preferred compounds of formula (IV) include those wherein Xaa₁ is His orAla. Preferably, Xaa₁ is His.

Preferred compounds of formula (IV) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (IV) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (IV) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (IV) include those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (IV) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (IV) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (IV) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (IV) include those wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from the group consisting ofPro, homoproline, thioproline and Methylalanine.

Preferred compounds of formula (IV) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (IV) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (IV) include those having an amino acidsequence described in PCT application Serial No. PCT/US98/24273, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as beingselected from SEQ. ID. NOS. 95-110 therein.

Formula V

Also provided are compounds described in PCT application PCT/US98/24210,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”,including compounds of the formula (V) [SEQ. ID. NO. 45]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅Xaa₂₆ X₁-Z₁; wherein

-   Xaa₁ is Ala, His, Arg or Tyr or 4-imidazopropionyl;-   Xaa₂ is Ser, Gly, Ala or Thr;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe, Tyr or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Ala, Asp or Glu;-   Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀    straight chain or branched alkanoyl or cycloalkylalkanoyl;-   Xaa₂₂ is Phe, Tyr or naphthylalanine;-   Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;-   Xaa₂₆ is Ala or Leu;-   X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R,    Lys-NH^(ε)—R Ala, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀    straight chain or branched alkanoyl or cycloalkylalkanoyl-   Z₁ is —OH,    -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;        wherein    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, and Xaa₂₆ are Ala. Also within the        scope of the present invention are pharmaceutically acceptable        salts of the compound of formula (V) and pharmaceutical        compositions including said compounds and salts thereof.

Preferred exendin agonist compounds of formula (V) include those whereinXaa₁ is His, Tyr or 4-imidazopropionyl. More preferably Xaa₁ is His.

Preferred are those compounds of formula (V) wherein Xaa₁ is4-imidazopropionyl.

Preferred are those compounds of formula (V) wherein Xaa₂ is Gly.

Preferred compounds of formula (V) are those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

According to one aspect, preferred are compounds of formula (V) whereinXaa₆ is Phe or naphthylalanine; and Xaa₂₂ is Phe or naphthylalanine; andXaa₂₃ is Ile or Val. More preferably, Z₁ is —NH₂. According to oneaspect, especially preferred are such compounds of formula (V) whereinXaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine. Morepreferred, Z₂ is —NH₂.

Preferred compounds of formula (V) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl. Preferred compounds of formula (V)include compounds described in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and identified therein as Compound Nos. 62-69.

Preferred are those compounds of formula (V) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (V) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (V) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (V) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (V) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (V) wherein: Xaa₁ is His or Ala; Xaa₂is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₅ is Ala or Thr; Xaa₆ is Pheor naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ isAla, Asp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala orSer; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu, Met orpentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ SerSer-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ orGly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ being independently Pro homoproline, thioproline orN-methylalanine; and Z₂ being —OH or —NH₂; provided that no more thanthree of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄,Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ andXaa₂₈ are Ala; and provided also that, if Xaa₁ is His, Arg or Tyr, thenat least one of Xaa₃, and Xaa₉ is Ala. Especially preferred compounds offormula (V) include those described in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and having the amino acid sequences identified therein asSEQ. ID. NOS. 5-93.

According to an especially preferred aspect, provided are compounds offormula (V) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula VI

Also provided are peptide compounds described in PCT Application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VI) [SEQ. ID. NO. 46]:

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅Xaa₂₆ X₁-Z₁; wherein

-   Xaa₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or    4imidazopropionyl;-   Xaa₂ is Ser, Gly, Ala or Thr;-   Xaa₃ is Ala, Asp or Glu;-   Xaa₄ is Ala, Norval, Val, Norleu or Gly;-   Xaa₅ is Ala or Thr;-   Xaa₆ is Ala, Phe, Tyr or naphthylalanine;-   Xaa₇ is Thr or Ser;-   Xaa₈ is Ala, Ser or Thr;-   Xaa₉ is Ala, Norval, Val, Norleu, Asp or Glu;-   Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;-   Xaa₁₁ is Ala or Ser;-   Xaa₁₂ is Ala or Lys;-   Xaa₁₃ is Ala or Gln;-   Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;-   Xaa₁₅ is Ala or Glu;-   Xaa₁₆ is Ala or Glu;-   Xaa₁₇ is Ala or Glu;-   Xaa₁₉ is Ala or Val;-   Xaa₂₀ is Ala or Arg;-   Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C¹⁻¹⁰    straight chain or branched alkanoyl or cycloalleyl-alkanoyl;-   Xaa₂₂ is Phe, Tyr or naphthylalanine;-   Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;-   Xaa₂₄ is Ala, Glu or Asp;-   Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;-   Xaa₂₆ is Ala or Leu;-   X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R,    Lys-NH^(ε)—R Ala, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀    straight chain or branched alkanoyl or cycloalkylalkanoyl-   Z₁ is —OH,    -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;    -   wherein    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine;    -   Xaa₃₁ is Ser or Tyr; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆,        Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,        Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, are Ala; and        provided also that, if Xaa₁ is His, Arg, Tyr, or        4-imidazopropionyl then at least one of Xaa₃, Xaa₄ and Xaa₉ is        Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VI) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (VI) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (VI) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (VI) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (VI) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VI) include those wherein Xaa₃₉ is Seror Tyr, preferably Ser.

Preferred compounds of formula (VI) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (VI) include those 42 wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₂₁ isLys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

Preferred compounds of formula (VI) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl.

Preferred compounds of formula (VI) include those described in PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” as having an amino acid sequenceselected from those identified therein as SEQ. ID. NOS. 95-110.

Formula VII

Compounds particularly useful according to the present invention areexendin agonist compounds described in U.S. patent application Ser. No.09/003,869, filed Jan. 7, 1998, entitled “Use of Exendins And AgonistsThereof For The Reduction of Food Intake”, including compounds of theformula (VII) [SEQ. ID. NO. 47]:

Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Ser Lys Gln Xaa₁₄ GluGlu Glu Ala Val Arg Leu Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Leu Lys Asn Gly GlyXaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z

wherein Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ isAsp or Glu; Xaa₆ is Phe, Tyr or naphthalanine; Xaa₇ is Thr or Ser; Xaa₈is Ser or Thr; Xaa₉ is Asp or Glu; Xaa₁₀ is Leu, Ile, Val, pentylglycineor Met; Xaa₁₄ is Leu, Ile, pentylglycine, Val or Met; Xaa₂₂ is Phe, Tyror naphthalanine; Xaa₂₃ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₂₄ is Glu or Asp; Xaa₂₅ is Trp, Phe, Tyr,or naphthylalanine; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₃₉ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either SEQ. ID. NOS. 1 or 2. Preferred N-alkyl groups forN-alkylglycine, N-alkylpentylglycine and N-alkylalanine include loweralkyl groups preferably of 1 to about 6 carbon atoms, more preferably of1 to 4 carbon atoms. Suitable compounds include those having amino acidsequences of SEQ. ID. NOS. 10 to 40. Also useful in the presentinvention are pharmaceutically acceptable salts of the compounds offormula (VII).

Preferred exendin agonist compounds include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycine orMet.

Preferred compounds include those wherein Xaa₂₅ is Trp or Phe.

Also preferred are compounds where Xaa₆ is Phe or naphthalanine; Xaa₂₃is Ile or Val and Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

According to an especially preferred aspect, Xaa₃₆, Xaa₃₇ and Xaa₃₈ arethe same amino acid reside.

Preferred are compounds wherein Xaa₃₉ is Ser or Tyr, more preferablySer.

Preferably Z is —NH₂.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ isPhe or naphthalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Pheor naphthalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (VII) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly; Xaa₃ is Asp or Glu; Xaa₆ is Phe or napthylalanine; Xaa₇ isThr or Ser; Xaa₈ is Ser or Thr; Xaa₉ is Asp or Glu; Xaa₁₀ is Leu orpentylglycine; Xaa₁₄ is Leu or pentylglycine; Xaa₂₂ is Phe ornaphthylalanine; Xaa₂₃ is Ile, Val or t-butyltylglycine; Xaa₂₄ is Glu orAsp; Xaa₂₅ is Trp or Phe; Xaa₃₁, Xaa₃₆, Xaa₃₇, and Xaa₃₈ areindependently Pro, homoproline, thioproline, or N-methylalanine; Xaa₃₉is Ser or Tyr: and Z is —OH or —NH₂; with the proviso that the compounddoes not have the formula of either SEQ. ID. NOS. 1 or 2. Morepreferably Z is —NH₂. Especially preferred compounds include thosehaving the amino acid sequence of SEQ. ID. NOS. 10, 11, 22, 23, 24, 27,29, 36, 37 and 40.

According to an especially preferred aspect, provided are compoundswhere Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds are believed to exhibitadvantageous duration of action and to be less subject to oxidativedegration, both in vitro and in vivo, as well as during synthesis of thecompound.

Formula VIII

Also provided are compounds described in PCT Application Serial No.PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VIII) [SEQ. ID. NO. 48]:

-   -   Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Ser Lys Gln        Xaa₁₄ Glu Glu Glu Ala Val Arg Leu Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Leu X₁        Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z        wherein Xaa₁ is His, Arg, Tyr or 4-imidazopropionyl; Xaa₂ is        Ser, Gly, Ala or Thr; Xaa₃ is Asp or Glu; Xaa₆ is Phe, Tyr or        naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ is Ser or Thr; Xaa₉ is        Asp or Glu; Xaa₁₀ is Leu, Ile, Val, pentylglycine or Met; Xaa₁₄        is Leu, Ile, pentylglycine, Val or Met; Xaa₂₂ is Phe, Tyr or        naphthylalanine; Xaa₂₃ is Ile, Val, Leu, pentylglycine,        tert-butylglycine or Met; Xaa₂₄ is Glu or Asp; Xaa₂₅ is Trp,        Phe, Tyr, or naphthylalanine; X₁ is Lys Asn, Asn Lys,        Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀        straight chain or branched alkanoyl or cycloalkylalkanoyl;        Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,        N-alkylpentylglycine or N-alkylalanine; Xaa₃₉ is Ser, Thr or        Tyr; and Z is —OH or —NH₂; with the proviso that the compound        does not have the formula of either SEQ. ID. NOS. 1 or 2.        Suitable compounds of formula (VIII) include compounds described        in PCT Application Serial No. PCT/US98/16387, filed Aug. 6,        1998, entitled “Novel Exendin Agonist Compounds” having the        amino acid sequences of SEQ. ID. NOS. 37-40 therein.

Preferred exendin agonist compounds of formula (VIII) include thosewherein Xaa₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa₁ isHis or 4-imidazopropionyl.

Preferred are those compounds of formula (VIII) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (VIII) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred are those compounds of formula (VIII) wherein Xaa₂₅ is Trp orPhe.

Preferred are those compounds of formula (VIII) wherein X₁ is Lys Asn,or Lys-NH^(ε)—R Asn, where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl.

Also preferred are compounds of formula (VIII) wherein Xaa₆ is Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Valand Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from Pro,homoproline, thioproline or N-alkylalanine. According to an especiallypreferred aspect, Xaa₃₉ is Ser or Tyr. Preferred are those suchcompounds wherein Xaa₃₉ is Ser. Preferably, Z is —NH₂.

According to one preferred aspect, preferred are compounds of formula(VIII) wherein Xaa₆ is Phe or naphthylalanine; Xaa₂₂ is Phe ornaphthylalanine; Xaa₂₃ is Ile or Val, X₁ is Lys Asn, or Lys-NH^(ε)—RAsn, where R is Lys, Arg, C₁-C₁₀ straight chain or branched alkanoyl andXaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from Pro,homoproline, thioproline or N-alkylalanine.

Preparation of Modified Exendins and Exendin Agonists

The modified exendins and exendin agonists of the present invention maybe made by linking one or more polyethylene glycol polymers to anexendin or exendin agonist. The synthesis of exendins and exendinagonists is thus described first, followed by methodology for linkingthe polyethylene glycol polymer(s) to the exendin or exendin agonist.

Preparation of Exendins and Exendin Agonists

Exendins and exendin agonist compounds such as exendin analogs andexendin derivatives, described herein may be prepared through peptidepurification as described in, for example, Eng, et al., J. Biol. Chem.265:20259-62, 1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992,hereby incorporated by reference herein. Alternatively, exendins andexendin agonist peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al. (J.Biol. Chem. 267:21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer. Thecompounds that constitute active ingredients of the formulations anddosages of the present invention may be prepared using standardsolid-phase peptide synthesis techniques and preferably an automated orsemiautomated peptide synthesizer. Typically, using such techniques, anα-N-carbamoyl protected amino acid and an amino acid attached to thegrowing peptide chain on a resin are coupled at room temperature in aninert solvent such as dimethylformamide, N-methylpyrrolidinone ormethylene chloride in the presence of coupling agents such asdicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of abase such as diisopropylethylamine. The α-N-carbamoyl protecting groupis removed from the resulting peptide-resin using a reagent such astrifluoroacetic acid or piperidine, and the coupling reaction repeatedwith the next desired N-protected amino acid to be added to the peptidechain. Suitable N-protecting groups are well known in the art, witht-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) beingpreferred herein.

The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.). The following side-chain protected aminoacids may be purchased from Applied Biosystems, Inc.:BSD-112344.1-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl),Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z),Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt),Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased fromApplied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole,dimethylsulfide, phenol, ethanedithiol, and thioanisole may be obtainedfrom Aldrich Chemical Company (Milwaukee, Wis.). Air Products andChemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic acid andmethanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).

Solid phase peptide synthesis may be carried out with an automaticpeptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry(see, Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resinsmay be cleaved with HF (−50° C. to 0° C., 1 hour). The peptide may beextracted from the resin with alternating water and acetic acid, and thefiltrates lyophilized. The Fmoc-peptide resins may be cleaved accordingto standard methods (Introduction to Cleavage Techniques, AppliedBiosystems, Inc., 1990, pp. 6-12). Peptides may also be assembled usingan Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).

Peptides may be purified by RP-HPLC (preparative and analytical) using aWaters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ,2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, andpurity may be determined using a C4, C8 or C18 analytical column (5μ,0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) maybe delivered to the analytical column at a flowrate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen, et al., The Pico Tag Method: A Manual of AdvancedTechniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation,Milford, Mass. (1989)). Fast atom bombardment analysis may be carriedout by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.Electrospray mass spectroscopy may be carried and on a VG-Trio machine.

Peptide active ingredient compounds useful in the formulations anddosages of the invention may also be prepared using recombinant DNAtechniques, using methods now known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor(1989). Alternatively, such compounds may be prepared by homogeneousphase peptide synthesis methods. Non-peptide compounds useful in thepresent invention may be prepared by art-known methods. For example,phosphate-containing amino acids and peptides containing such aminoacids, may be prepared using methods known in the art. See, e.g.,Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).

Conjugation of Polyethylene Glycol Polymers

There are several strategies for coupling PEG to peptides/proteins. See,Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); andCrit. Rev. Therap. Drug Carrier Sys. 9:249 (1992) all of which areincorporated herein by reference in their entirety. Those skilled in theart, therefore, will be able to utilize such well-known techniques forlinking one or more polethylene glycol polymers to the exendins andexendin agonists described herein. Suitable polethylene glycol polymerstypically are commercially available or may be made by techniques wellknow to those skilled in the art. The polyethylene glycol polymerspreferably have molecular weights between 500 and 20,000 and may bebranched or straight chain polymers.

The attachment of a PEG on an intact peptide or protein can beaccomplished by coupling to amino, carboxyl or thiol groups. Thesegroups will typically be the N and C termini and on the side chains ofsuch naturally occurring amino acids as lysine, aspartic acid, glutamicacid and cysteine. Since exendin 4 and other exendins and exendinagonists can be prepared by solid phase peptide chemistry techniques, avariety of moieties containing diamino and dicarboxylic groups withorthogonal protecting groups can be introduced for conjugation to PEG.

The present invention also provides for conjugation of an exendin orexendin agonist to one or more polymers other than polyethylene glycolwhich can regulate kidney clearance in a manner similar to polyethyleneglycol. Examples of such polymers include albumin and gelatin. See,Gombotz and Pettit, Bioconjugate Chem., 6:332-351, 1995, which isincorporated herein by reference in its entirety.

Utility

The formulations and dosages described herein are useful in view oftheir pharmacological properties. In particular, the compounds describedherein possess activity as agents to reduce glucagon levels and suppressglucagon secretion, as evidenced by the ability to lower glucagon levelsin animals and humans. They can be used to treat conditions or diseasesthat can be alleviated by reducing glucagon levels and suppressingglucagon secretion.

The compounds referenced above may form salts with various inorganic andorganic acids and bases. Such salts include salts prepared with organicand inorganic acids, for example, HCl, HBr, H₂SO₄, H₃PO₄,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid,toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonicacid. Salts prepared with bases include ammonium salts, alkali metalsalts, e.g., sodium and potassium salts, and alkali earth salts, e.g.,calcium and magnesium salts. Acetate, hydrochloride, andtrifluoroacetate salts are preferred. The salts may be formed byconventional means, as by reacting the free acid or base forms of theproduct with one or more equivalents of the appropriate base or acid ina solvent or medium in which the salt is insoluble, or in a solvent suchas water which is then removed in vacuo or by freeze-drying or byexchanging the ions of an existing salt for another ion on a suitableion exchange resin.

Formulation and Administration

Exendin and exendin agonist formulations and dosages of the inventionare useful in view of their exendin-like effects, and may convenientlybe provided in the form of formulations suitable for parenteral(including intravenous, intramuscular and subcutaneous) administration.Also described herein are formulations and dosages useful in alternativedelivery routes, including oral, nasal, buccal, sublingual andpulmonary.

The feasibility of alternate routes of delivery for exendin-4 has beenexplored by measuring exendin-4 in the circulation in conjunction withobservation of a biologic response, such as plasma glucose lowering indiabetic animals, after administration. Passage of exendin-4 has beeninvestigated across several surfaces, the respiratory tract (nasal,tracheal and pulmonary routes) and the gut (sublingual, gavage andintraduodenal routes). Biologic effect and appearance of exendin-4 inblood have been observed with each route of administration via therespiratory tract, and with sublingual and gavaged peptide via thegastrointestinal tract. Intra-tracheal administration, nasaladministration, administration via the gut, and sublingualadministration have all been described.

In some cases, it will be convenient to provide a modified exendin orexendin agonist and another anti-glucagon agent, such as an amylin or anamylin agonist, in a single composition or solution for administrationtogether. In other cases, it may be more advantageous to administeranother anti-glucagon agent separately from the exendin, exendinagonist, or modified exendin or exendin agonist. In yet other cases, itmay be beneficial to provide an exendin, exendin agonist, or modifiedexendin or exendin agonist either co-formulated or separately with otherglucagon lowering agents such as amylin. A suitable administrationformat may best be determined by a medical practitioner for each patientindividually. Suitable pharmaceutically acceptable carriers and theirformulation are described in standard formulation treatises, e.g.,Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y.J. and Hanson, M. A. “Parenteral Formulations of Proteins and Peptides:Stability and Stabilizers,” Journal of Parenteral Science andTechnology, Technical Report No. 10, Supp. 42:2S (1988).

Compounds useful in the invention can be provided as parenteralcompositions for injection or infusion. They can, for example, besuspended in an inert oil, suitably a vegetable oil such as sesame,peanut, olive oil, or other acceptable carrier. Preferably, they aresuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 3.0 to about 7.0, more specifically from about4.0 to 6.0, and preferably from about 4.0 to about 5.0. Thesecompositions may be sterilized by conventional sterilization techniques,or may be sterile filtered. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH buffering agents.Useful buffers include for example, sodium acetate/acetic acid buffers.A form of repository or “depot” slow release preparation may be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery.

The desired isotonicity may be accomplished using sodium chloride orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. Sodium chloride ispreferred particularly for buffers containing sodium ions.

The claimed compounds can also be formulated as pharmaceuticallyacceptable salts (e.g., acid addition salts) and/or complexes thereof.Pharmaceutically acceptable salts are non-toxic salts at theconcentration at which they are administered. The preparation of suchsalts can facilitate the pharmacological use by altering thephysical-chemical characteristics of the composition without preventingthe composition from exerting its physiological effect. Examples ofuseful alterations in physical properties include lowering the meltingpoint to facilitate transmucosal administration and increasing thesolubility to facilitate the administration of higher concentrations ofthe drug.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, phosphate, sulfamate, acetate,citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.Pharmaceutically acceptable salts can be obtained from acids such ashydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, aceticacid, citric acid, lactic acid, tartaric acid, malonic acid,methanesulfonic acid, ethane-sulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Suchsalts may be prepared by, for example, reacting the free acid or baseforms of the product with one or more equivalents of the appropriatebase or acid in a solvent or medium in which the salt is insoluble, orin a solvent such as water which is then removed in vacuo or byfreeze-drying or by exchanging the ions of an existing salt for anotherion on a suitable ion exchange resin.

Carriers or excipients can also be used to facilitate administration ofthe compound. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents. The compositions or pharmaceutical composition can beadministered by different routes including intravenously,intraperitoneal, subcutaneous, and intramuscular, orally, topically, ortransmucosally.

If desired, solutions of the above compositions may be thickened with athickening agent such as methylcellulose. They may be prepared inemulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

Compositions useful in the invention are prepared by mixing theingredients following generally accepted procedures. For example, theselected components may be simply mixed in a blender or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity.

Other pharmaceutically acceptable carriers and their formulation aredescribed in standard formulation treatises, e.g., Remington'sPharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. andHanson, M. A. “Parenteral Formulations of Proteins and Peptides:Stability and Stabilizers,” Journal of Parenteral Science andTechnology, Technical Report No. 10, Supp. 42:2S (1988).

For use by the physician, the compounds will be provided in dosage unitform containing an amount of an exendin, exendin agonist, or modifiedexendin or exendin agonist, with or without another anti-glucagon agent.Therapeutically effective amounts of an exendin, exendin agonist, ormodified exendin or exendin agonist for use in the control of glucagonand in conditions in which glucagon levels are beneficially lowered orregulated are those that decrease post-prandial blood glucagon levels asdesired. In diabetic or glucose intolerant individuals, plasma glucagonlevels may be higher than in normal individuals. In such individuals,beneficial reduction or “smoothing” of post-prandial blood glucagonlevels, may be obtained. As will be recognized by those in the field, aneffective amount of therapeutic agent will vary with many factorsincluding the age and weight of the patient, the patient's physicalcondition, the glucagon level or level of inhibition of glucagonsuppression to be obtained, and other factors.

Such pharmaceutical compositions are useful in causing glucagon to belowered in a subject and may be used as well in other disorders wherelowered or suppressed glucagon is beneficially reduced.

The effective daily anti-glucagon dose of the compounds will typicallybe in the range of 0.01 or 0.03 to about 5 mg/day, preferably about 0.01or 0.5 to 2 mg/day and more preferably about 0.01 or 0.1 to 1 mg/day,for a 70 kg patient, administered in a single or divided doses. Theexact dose to be administered is determined by the attending clinicianand is dependent upon where the particular compound lies within theabove quoted range, as well as upon the age, weight and condition of theindividual. Administration should begin at the first sign of symptoms orshortly after diagnosis of, for example, diabetes mellitus as manifestedby elevated glucagon. Administration may be by injection, preferablysubcutaneous or intramuscular. Orally active compounds may be takenorally, however dosages should be increased 5-10 fold.

Generally, in treating or preventing elevated, inappropriate, orundesired post-prandial blood glucagon levels, the compounds of thisinvention may be administered to patients in need of such treatment in adosage ranges similar to those given above, however, the compounds areadministered more frequently, for example, one, two, or three times aday. Particularly preferred are the exendin and exendin agonistformulations and dosages and routes of administration thereof describedcommonly owned U.S. Provisional Application 60/116,380, entitled “NovelExendin Agonist Formulations And Methods Of Administration Thereof,”filed Jan. 14, 1999 (and the corresponding PCT application claimingpriority from it that was filed on Jan. 14, 2000, Ser. No. ______, andU.S. Provisional Application 60/______, entitled “Use of Exendins andAgonists Thereof for Modulation of Triglyceride Levels and Treatment ofDyslipidemia,” filed Jan. 14, 1999, from which this application claimspriority and the disclosures of which have been incorporated byreferenced in their entirety as if fully set forth herein.

A form of repository or “depot” slow release preparation may be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or other form of delivery.

The effective daily doses of the compounds are described. The exact doseto be administered may be determined by the attending clinician and maybe further dependent upon the efficacy of the particular exendin orexendin agonist compound used, as well as upon the age, weight andcondition of the individual. The optimal mode of administration ofcompounds of the present application to a patient depend on factorsknown in the art such as the particular disease or disorder, the desiredeffect, and the type of patient. While the compounds will typically beused to treat human patients, they may also be used to treat similar oridentical diseases in other vertebrates such as other primates, farmanimals such as swine, cattle and poultry, and sports animals and petssuch as horses, dogs and cats.

In particular, the formulation which best supports a parenteral liquiddosage form is one in which the active ingredient(s) is stable withadequate buffering capacity to maintain the pH of the solution over theintended shelf life of the product. The dosage form should be either anisotonic and/or an iso-osmolar solution to either facilitate stabilityof the active ingredient or lessen the pain on injection or both.Devices that deliver very small injection volumes, however, may notrequire that the formulation be either isotonic and/or iso-osmolar. Ifthe dosage form is packaged as a unit-dose, then a preservative may beincluded but is not required. If, however, the dosage form is packagedin a multi-use container, then a preservative is necessary.

These dosage forms include approximately 0.005 to about 0.4%, morespecifically from about 0.005 to about 0.02%, or from about 0.005 toabout 0.05% (w/v), respectively of the active ingredient in an aqueoussystem along with approximately 0.02 to 0.5% (w/v) of an acetate,phosphate, citrate or glutamate or similar buffer either alone or incombination to obtain a pH of the final composition of approximately 3.0to 7.0, more specifically from about pH 4.0 to about 6.0, or from about4.0 to 5.0, as well as either approximately 1.0 to 10% (w/v) of acarbohydrate or polyhydric alcohol iso-osmolality modifier (preferablymannitol) or up to about 0.9% saline or a combination of both leading toan isotonic or an iso-osmolar solution in an aqueous continuous phase.Approximately 0.005 to 1.0% (w/v) of an anti-microbial preservativeselected from the group consisting of m-cresol, benzyl alcohol, methylethyl, propyl and butyl parabens and phenol is also present if theformulation is packaged in a multi-use container. A sufficient amount ofwater for injection is added to obtain the desired concentration ofsolution. Sodium chloride, as well as other excipients, may also bepresent, if desired. Such excipients, however, must maintain the overallstability of the active ingredient.

Polyhydric alcohols and carbohydrates share the same feature in theirbackbones, i.e., —CHOH—CHOH—. The polyhydric alcohols include suchcompounds as sorbitol, mannitol, glycerol, and polyethylene glycols(PEGs). These compounds are straight-chain molecules. The carbohydrates,such as mannose, ribose, trehalose, maltose, glycerol, inositol, glucoseand lactose, on the other hand, are cyclic molecules that may contain aketo or aldehyde group. These two classes of compounds will also beeffective in stabilizing protein against denaturation caused by elevatedtemperature and by freeze-thaw or freeze-drying processes. Suitablecarbohydrates include galactose, arabinose, lactose or any othercarbohydrate which does not have an adverse affect on a diabeticpatient, i.e., the carbohydrate is not metabolized to form largeconcentrations of glucose in the blood. Such carbohydrates are wellknown in the art as suitable for diabetics.

Preferably, the peptides of the present invention are admixed with apolyhydric alcohol such as sorbitol, mannitol, inositol, glycerol,xylitol, and polypropylene/ethylene glycol copolymer, as well as variouspolyethylene glycols (PEG) of molecular weight 200, 400, 1450, 3350,4000, 6000, and 8000). Mannitol is the preferred polyhydric alcohol.

The liquid formulation of the invention should be substantially isotonicand/or iso-osmolar. An isotonic solution may be defined as a solutionthat has a concentration of electrolytes, or a combination ofelectrolytes and non-electrolytes that will exert equivalent osmoticpressure as that into which it is being introduced, here for example inthe case of parenteral injection of the formulation, a mammalian tissue.Similarly, an iso-osmolar solution may be defined as a solution that hasa concentration of non-electrolytes that will exert equivalent osmoticpressure as that into which it is being introduced. As used herein,“substantially isotonic” means within ±20% of isotonicity, preferablywithin +10%. As used herein, “substantially iso-osmolar” means within+20% of iso-osmolality, preferably within ±10%. The formulated productfor injection is included within a container, typically, for example, avial, cartridge, prefilled syringe or disposable pen.

The formulation which best support a unit-dose parenteral lyophilizeddosage form is one in which the active ingredient is reasonably stable,with or without adequate buffering capacity to maintain the pH of thesolution over the intended shelf life of the reconstituted product. Thedosage form should contain a bulking agent to facilitate cake formation.The bulking agent may also act as a tonicifer and/or iso-osmolalitymodifier upon reconstitution to either facilitate stability of theactive ingredient and/or lessen the pain on injection. As noted above,devices that deliver very small injection volumes may not require theformulation to be isotonic and/or iso-osmolar. A surfactant may alsobenefit the properties of the cake and/or facilitate reconstitution.

These dosage forms include approximately 0.005 to about 0.4%, morespecifically from about 0.005 to about 0.02%, or 0.005 to 0.05% (w/v) ofthe active ingredient. It may not be necessary to include a buffer inthe formulation and/or to reconstitute the lyophile with a buffer if theintention is to consume the contents of the container within thestability period established for the reconstituted active ingredient. Ifa buffer is used, it may be included in the lyophile or in thereconstitution solvent. Therefore, the formulation and/or thereconstitution solvent may contain individually or collectivelyapproximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate orglutamate buffer either alone or in combination to obtain a pH of thefinal composition of approximately 3.0 to 7.0, more specifically fromabout pH 4.0 to about 6.0, or from about 4.0 to 5.0. The bulking agentmay consist of either approximately 1.0 to 10% (w/v) of a carbohydrateor polyhydric alcohol iso-osmolality modifier (as described above) or upto 0.9% saline or a combination of both leading to a isotonic oriso-osmolar solution in the reconstituted aqueous phase. A surfactant,preferably about 0.1 to about 1.0% (w/v) of polysorbate 80 or othernon-ionic detergent, may be included. As noted above, sodium chloride,as well as other excipients, may also be present in the lyophilizedunit-dosage formulation, if desired. Such excipients, however, mustmaintain the overall stability of the active ingredient. The formulationwill be lyophilized within the validation parameters identified tomaintain stability of the active ingredient.

The liquid formulation of the invention before lyophilization should besubstantially isotonic and/or iso-osmolar either before lyophilizationor to enable formation of isotonic and/or iso-osmolar solutions afterreconstitution. The formulation should be used within the periodestablished by shelf-life studies on both the lyophilized form andfollowing reconstitution. The lyophilized product is included within acontainer, typically, for example, a vial. If other containers are usedsuch as a cartridge, pre-filled syringe, or disposable pen, thereconstitution solvent may also be included.

As with the parenteral liquid and lyophilized unit-dosage formulationsdescribed above, the formulation which best supports a multi-doseparenteral lyophilized dosage form is one in which the active ingredientis reasonably stable with adequate buffering capacity to maintain the pHof the solution over the intended “in-use” shelf-life of the product.The dosage form should contain a bulking agent to facilitate cakeformation. The bulking agent may also act as a tonicifer and/oriso-osmolality modifier upon reconstitution to either facilitatestability of the active ingredient or lessen the pain on injection orboth. Again, devices that deliver very small injection volumes may notrequire the formulation to be either isotonic and/or iso-osmolar. Apreservative is, however, necessary to facilitate multiple use by thepatient.

These dosage forms include approximately 0.005 to about 0.4%, morespecifically from about 0.005 to about 0.02%, or from about 0.005 to0.05% (w/v), respectively of the active ingredient. It may not benecessary to include a buffer in the formulation and/or to reconstitutethe lyophile with a buffer if the intention is to consume the contentsof the container within the stability period established for thereconstituted active ingredient. If a buffer is used, it may be includedin the lyophile or in the reconstitution solvent. Therefore, theformulation and/or the reconstitution solvent may contain individuallyor collectively approximately 0.02 to 0.5% (w/v) of an acetate,phosphate, citrate or glutamate buffer either alone or in combination toobtain a pH of the final composition of approximately 3.0 to 7.0, morespecifically from about pH 4.0 to about 6.0, or from about 4.0 to 5.0.The bulking agent may consist of either approximately 1.0 to 10% (w/v)of a carbohydrate or a polyhydric alcohol iso-osmolality modifier(preferably mannitol) or up to 0.9% saline, or a combination of both,leading to an isotonic or iso-osmolar solution in the reconstitutedaqueous phase. A surfactant, preferably about 0.1 to about 1.0% (w/v) ofpolysorbate 80 or other non-ionic detergent, may be included.Approximately 0.005 to 1.0% (w/v) of an anti-microbial preservativeselected from the group consisting of m-cresol, benzyl alcohol, methyl,ethyl, propyl and butyl parabens and phenol (preferably m-cresol) isalso present if the formulation is packaged in a multi-use container.Sodium chloride, as well as other excipients, may also be present, ifdesired. Again, however, such excipients must maintain the overallstability of the active ingredient. The formulation should belyophilized within the validation parameters identified to maintainstability of the active ingredient. The liquid formulation of theinvention should be substantially isotonic and/or iso-osmolar eitherbefore lyophilization or to enable formation of isotonic and/oriso-osmolar solutions after reconstitution. The formulation should beused within the period established by shelf-life studies on both thelyophilized form and following reconstitution. The lyophilized productis included within a container, typically, for example, a vial. If othercontainers are used such as a cartridge, pre-filled syringe ordisposable pen, the reconstitution solvent may also be included.

The formulations that best support oral, nasal, pulmonary and/orintra-tracheal dosage forms may be either preserved or unpreservedliquid formulations and/or dry powder or, for oral administration, solidformulations. The preserved or unpreserved liquid formulations will beessentially identical to the formulations described above underpreserved or unpreserved liquid parenteral formulations. The pH of thesolution should be about 3.0 to 7.0, with a pH greater than or equal toabout 5.0 being most preferred to reduce the potential forbronchoconstriction. The dry powder formulations may contain a bulkingagent and/or salts to facilitate particle size formation and appropriateparticle size distribution. A surfactant and/or salts may also benefitthe properties of the particle morphology and/or facilitate tissueuptake of the active ingredient.

These dry powder dosage forms can range from 1% to 100% (w/w),respectively of the active ingredient. It may not be necessary toinclude a bulking agent and/or salts to facilitate particle sizeformation and/or distribution. The bulking agent and/or salts mayconsist of either approximately 0 to 99% (w/w) of a carbohydrate orpolyhydric alcohol or approximately 0 to 99% salt or a combination ofboth leading to the preferred particle size and distribution. Asurfactant, preferably about 0.1 to about 1.0% (w/w) of polysorbate 80or other non-ionic detergent, may be included. Sodium chloride, as wellas other excipients, may also be present, if desired. Such excipients,however, must maintain the overall stability of the active ingredientand facilitate the proper level of hydration.

The formulations that best support nasal and/or intra-tracheal dosageforms may be either preserved or unpreserved liquid dosage formulationsdescribed previously.

Dissolvable gels and/or patches may be used to facilitate buccaldelivery. The gels may be prepared from various types of starch and/orcellulose derivatives.

Sublingual delivery may be best supported by liquid formulations similarto those described above as parenteral liquid or parenteral lyophilizedformulations after reconstitution except without the need for the dosageform to be an isotonic and/or iso-osmolar solution. Solid dosage formsmay be similar to oral solid dosage forms except that they must bereadily dissolvable under the tongue.

Oral delivery may be best supported by a liquid (gel cap) formulationthat is similar to the parenteral liquid formulation except that thesolution may be more concentrated and may contain additional additivesto facilitate uptake of the active ingredient by the small intestine.Solid dosage forms will contain inert ingredients along with the activeingredient to facilitate tablet formation. These ingredients may includepolyhedral alcohols (such as mannitol), carbohydrates, or types ofstarch, cellulose derivatives, and/or other inert, physiologicallycompatible materials. The tablet may be enterically coated to minimizedigestion in the stomach and thereby facilitate dissolution and uptakefurther along the alimentary canal.

Clinical Studies

As described in Example 14 below, a double blind, placebo-controlledsingle ascending dose study examining the safety, tolerability, andpharmacokinetics of subcutaneous exendin-4 in healthy volunteers hasbeen completed. Five single subcutaneous doses of exendin-4 (0.01, 0.05,0.1, 0.2 or 0.3 μg/kg) were studied in 40 healthy male volunteers in thefasting state. Maximum plasma exendin-4 concentrations were achievedbetween one and two hours post-dose with little difference among thedoses examined. Examination of the data indicated a dose dependentincrease for C_(max). There were no serious adverse events reported inthis study.

In the healthy male volunteers that participated in this study,exendin-4 was well tolerated at subcutaneous doses up to and including0.1 μg/kg. A decrease in plasma glucose concentration was also observedat this dose. At doses of 0.2 μg/kg and higher, the most commonlyobserved adverse events were headache, nausea, vomiting, dizziness, andpostural hypotension. There was a transient fall in plasma glucoseconcentration following administration of doses of 0.05 μg/kg and above.

Alternate Routes of Delivery

The feasibility of alternate routes of delivery for exendin-4 has beenexplored by measuring exendin-4 in the circulation in conjunction withobservation of a biologic response, such as plasma glucose lowering indiabetic animals, after administration. Passage of exendin-4 has beeninvestigated across several surfaces, the respiratory tract (nasal,tracheal, and pulmonary routes) and the gut (sublingual, gavage andintraduodenal routes). Biologic effect and appearance of exendin-4 inblood have been observed with each route of administration via therespiratory tract, and with sublingual and gavaged peptide via thegastrointestinal tract.

Intra-tracheal Administration—As described herein, intra-trachealadministration of exendin-4 into fasted rats (20 μg/50 μL/animal)produced a rise in the mean plasma exendin-4 concentration to 2060±960pg/mL within 5-10 minutes after administration. Elevated plasmaexendin-4 concentrations were maintained for at least 1 hour afterinstillation (see FIG. 7). In diabetic db/db mice, intra-trachealinstillation of exendin-4 (1 μg/animal) lowered plasma glucoseconcentration by 30% while that in the vehicle control group increasedby 41% 1.5 hours after treatment. In these animals the mean plasmaconcentration of exendin-4 was 777±365 pg/ml at 4.5 hours aftertreatment (see FIGS. 8 a and 8 b).

In diabetic ob/ob mice, intra-tracheal instillation of exendin-4 (1μg/animal) decreased plasma glucose concentration to 43% of thepre-treatment level after 4 hours while that in the vehicle controlgroup was not changed (see FIGS. 9 a and 9 b).

Nine overnight-fasted male Sprague Dawley rats (age 96-115 days, weight365-395, mean 385 g) were anesthetized with halothane, tracheotomized,and catheterized via the femoral artery. At t=0 min, 30 μL of saline inwhich was dissolved 2.1 μg (n=3), 21 μg (n=3) or 210 μg of exendin-4 wasinstilled into the trachea beyond the level of intubation. Blood sampleswere taken after 5, 10, 20, 30, 60, 90, 120, 150, 180, 240, 300 and 360min, centrifuged and plasma stored at −20° C. for subsequentimmunoradiometric (IRMA) assay directed to N-terminal and C-terminalepitopes of the intact exendin-4 molecule. Following intra-trachealadministration, 61-74% of peak plasma concentration was observed within5 min. Tmax occurred between 20 and 30 min after administration. AUC andCmax were proportional to dose. At a dose of 2.1 μg (1.5 nmol/kg),resulting in plasma concentrations of ˜50 pM (where glucose-loweringeffects in man are observed), bioavailability was 7.3%. The coefficientof variation was 44%. At higher doses, bioavailability was slightlylower, and the CV was higher (see FIGS. 10 a and 10 b). Via the trachealroute of administration, the t % (defined pragmatically as time forplasma to fall below 50% of Cmax) was 30-60 min for the lowest dose and60-90 min for the 2 higher doses. In summary, biologically effectivequantities of exendin-4 are rapidly absorbed via the trachea withoutevoking apparent respiratory distress. The respiratory tract is a viableroute of administration of exendin-4.

Pulmonary Administration—Increased plasma concentrations of exendin-4were detected in rats exposed to aerosolized exendin-4. Exposure of ratsto approximately 8 ng of aerosolized exendin-4 per mL of atmosphere for10 minutes resulted in peak plasma exendin-4 concentrations of 300-1900pg/mL 5 minutes following treatment (see FIG. 11). Similar exposure ofdiabetic db/db mice to aerosolized exendin-4 lead to a 33% decrease inplasma glucose concentration after 1 hour, when a mean plasma exendin-4concentration of 170±67 pg/mL was detected. Diabetic db/db mice in thecontrol group exposed to aerosolized saline recorded no change in plasmaglucose (see FIGS. 12 a and 12 b).

Nasal administration—Application of exendin-4 into the nasal cavity ofrats led to a rise in plasma concentrations. Peak values of 300 pg/mLand 6757 pg/mL were detected 10 minutes after administration of 1 μg and100 μg exendin-4 (dissolved in 2 μL saline), respectively (see FIG. 13).

Administration via the Gut—Male db/db mice (approximately 50 g body wt.)were fasted for 2 h and before and after an intra-gastric administrationof saline or exendin-4 (exendin-4). A 9% decrease in plasma glucoseconcentration was observed with 1 mg/200 μl/animal and a 15% decreasewas observed with 3 mg/200 μl/animal, compared with a 10% increaseplasma glucose in the controls one hour after treatment (see FIG. 14).

Sublingual Administration—Sublingual application of exendin-4 (100 μg/5μL/animal) to diabetic db/db mice led to a 15% decrease in plasmaglucose concentration one hour after treatment. A 30% increase wasobserved for the control group receiving saline. The mean exendin-4plasma level at 60 minutes was 4520±1846 pg/mL (see FIGS. 15 a, 15 b,and 15 c).

Eight Sprague Dawley rats (˜300 g) were briefly anesthetized withmetophane while a solution containing 10 μg/3 μL (n=4) or 100 μg/3 μL(n=4) was pipetted under the tongue. Blood samples were subsequentlycollected from the topically anesthetized tail and assayed for exendin-4by IRMA. Plasma concentrations had begun to rise by 3 min afteradministration and were maximal 10 min and 30 min after administration(10 μg and 100 μg doses, respectively). Plasma exendin-4 concentrationsubsequently remained above the lower limit of quantitation (LLOQ)beyond 5 hours. Area-under-the-curve to the end of each experiment wascalculated by the trapezoidal method. Two numbers were derived, onederived from total immunoreactivity, the other derived from theincrement above the non-zero value present at t=0. These values werecompared to historical intravenous bolus data in the same animal modelto obtain, respectively, high and low estimates of bioavailability. Forthe 10 μg dose, sublingual bioavailability was 3.1-9.6%, and for a 100μg dose, bioavailability was lower at 1.3-1.5%. Variability of AUC wasgreatest in the first hour after administration (CV 74% and 128% for 10and 100 μg doses). For the 5-hour integral, coefficient of variation ofthe AUC was 20% and 64%, respectively. Peak plasma concentration (Cmax)occurred as rapidly after sublingual administration as aftersubcutaneous administration (Tmax ˜30 min). Cmax after sublingualadministration of 10 μg exendin-4 was 1.5% that after an intravenousbolus, but 14.5% of that obtained after a subcutaneous bolus. Cmax aftersublingual administration of 100 μg exendin-4 was only 0.29% of thatobserved after an intravenous bolus, and 6.1% of that obtained after asubcutaneous bolus (see FIGS. 15 d and 15 e). Thus, exendin-4 can bedelivered at bioeffective doses via the sublingual route.Bioavailability and C_(max) were greatest, T_(max) was shortest, andvariability of availability was least with the lowest sublingual dose.The lowest sublingual dose resulted in plasma concentrations similar tothose that are predicted to be effective in lowering glucose in humans(˜50-100 pM).

The optimal formulation and mode of administration of compounds of thepresent application to a patient depend on factors known in the art suchas the particular disease or disorder, the desired effect, and the typeof patient. While the compounds will typically be used to treat humanpatients, they may also be used to treat similar or identical diseasesin other vertebrates such as other primates, farm animals such as swine,cattle and poultry, and sports animals and pets such as horses, dogs andcats.

To assist in understanding the present invention the following Examplesare included which describe the results of a series of experiments. Theexperiments relating to this invention should not, of course, beconstrued as specifically limiting the invention and such variations ofthe invention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

EXAMPLE 1 Preparation of Exendin-3

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser-NH₂ [SEQ. ID. NO. 1]

The above amidated peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.). In general, single-coupling cycles were usedthroughout the synthesis and Fast Moc (HBTU activation) chemistry wasemployed. Deprotection (Fmoc group removal) of the growing peptide chainwas achieved using piperidine. Final deprotection of the completedpeptide resin was achieved using a mixture of triethylsilane (0.2 mL),ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) andtrifluoroacetic acid (15 mL) according to standard methods (Introductionto Cleavage Techniques, Applied Biosystems, Inc.) The peptide wasprecipitated in ether/water (50 mL) and centrifuged. The precipitate wasreconstituted in glacial acetic acid and lyophilized. The lyophilizedpeptide was dissolved in water). Crude purity was about 75%.

Used in purification steps and analysis were Solvent A (0.1% TFA inwater) and Solvent B (0.1% TFA in ACN).

The solution containing peptide was applied to a preparative C-18 columnand purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purityof fractions was determined isocratically using a C-18 analyticalcolumn. Pure fractions were pooled furnishing the above-identifiedpeptide. Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide gave product peptide havingan observed retention time of 19.2 minutes.

EXAMPLE 2 Preparation of Exendin-4

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser-NH₂ [SEQ. ID. NO. 2]

The above amidated peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Exendin-3 as describe in Example 1. Used inanalysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide gave product peptide havingan observed retention time of 14.9 minutes. Electrospray MassSpectrometry (M): calculated 4186.6; found 4186.0 to 4186.8 (four lots).

EXAMPLE 3 Clearance by the Kidney

The kidney can play a major role in the elimination of some molecules(drugs, peptides, proteins). For some molecules, this process beginswhen the kidney filters the blood at the glomerulus to produce theultrafiltrate described below. The glomerular filter discriminates notonly on the basis of molecular weight but also by acting as a negativelycharged selective barrier, promoting retention of anionic compounds. Thefree fraction of molecules in the plasma (not protein bound) with amolecular weight less than 5 kD and an effective radii less than 15 Åare easily filtered. For larger molecular weight molecules they arefiltered on a more restrictive and limited basis, principally bymolecular size, structure and net charge. The cutoff point forglomerular filtration lies between albumin (67 kD) which is retained andhemoglobin (68 kD) which is filtered. Albumin, with an effective radiusof about 36 Å is filtered less than 1% at the glomerulus.

Once in the glomerulus a molecule travels to the proximal tubule whereit is either reabsorbed or it passes on through the loop of Henle to thedistal tubule where collecting ducts drain the filtrate into thebladder. Filtered proteins and peptides are typically cleaved by brushborder enzymes in the proximal tubule, from where they are efficientlyretrieved by sodium/amino cotransporters (scavenging pumps). Otherwise,molecules which are polar, ionized and of large molecular weight willnot be reabsorbed. Throughout this process metabolizing enzymes in therenal cortex (proximal tubules) may also degrade the molecule into morepolar molecules, thereby increasing the probability for excretion intothe urine. Many peptide hormones (for example, amylin, calcitonins) aredegraded by passage through the renal circulation, presumably byvascular ectoenzymes accessible to the plasma, independently of theprocess of glomerular filtration. In those examples, rates of peptideclearance from the plasma are similar to the rate of renal plasma flow,which is ˜3-fold greater than the rate of glomerular filtration.

Studies performed to identify plasma circulating metabolites ofexendin-4 yielded very little evidence of proteolytic degradation;following large intravenous doses in animals, HPLC analysis of plasmashowed only the presence of intact exendin, and negligible appearance of“daughter” peaks indicative of the buildup of degradation products. Thisis in contrast to other peptides studied (for example amylin and GLP-1)where the disappearance of the “parent” HPLC peak was associated withthe appearance of “daughter” HPLC peaks, subsequently identified assubpeptide degradants. The absence of plasma degradants of exendinindicates little or no proteolysis at any site, including the renalcirculation. Any clearance by the kidney, then, is via non-proteolyticmeans, namely filtration or active excretion (as occurs with para-aminohippurate).

Initial measurements of exendin clearance in man (120-130 mL/min),monkeys (˜9 mL/min) and rats (3.2-4.4 mL/min) matched reportedglomerular filtration rates in those species. To test whether renalfiltration could be the principal mode of exendin elimination, studieswere performed in overnight fasted nephrectomized male rats infused withexendin at a constant rate. Steady-state plasma levels of exendin-4 weregreatly increased in nephrectomized rats compared to rats with theirkidneys intact. This data indicated that the kidney was responsible forat least 80% of the clearance of exendin 4 (see FIGS. 5 and 6). Exendinclearance rates in intact rats were, again, similar to glomerularfiltration rates expected in those rats (4.2 mL/min). Taken togetherthese results indicate that very little metabolism occurs systemicallyand that most of the clearance of exendin 4 is through the kidney viafiltration (but not by renal intravascular proteolysis). The low amountsof immunoreactive full-length exendin in the urine are consistent withit being cleaved by brush border enzymes in the proximal tubule afterfiltration.

EXAMPLE 4 Exendin-4 Decreases Glucagon Secretion During HyperglycemicClamps in Diabetic Fatty Zucker Rats

Absolute or relative hyperglucagonemia is often a feature of, forexample, type 1 and type 2 diabetes mellitus, and the suppression ofexcessive glucagon secretion in these and other conditions described orreferred to herein is a potential benefit of therapy usingglucagonostatic agents. In this Example, the effect of exendin-4 onglucagon secretion in male anaesthetized Diabetic Fatty Zucker (ZDF)rats was examined. Using an hyperinsulinemic hyperglycemic clampprotocol, factors tending to influence glucagon secretion were heldconstant. Plasma glucose was clamped at −34 mM 60 min before beginningintravenous infusions of saline (n=7) or exendin-4 (0.21 μg+2.1 μg/mL/h;n=7). Plasma glucagon concentration measured prior to these infusionswere similar in both groups (306±30 pM versus 252±32 pM, respectively;n.s.).

Mean plasma glucagon concentration in exendin-4 infused rats was nearlyhalf of that in saline-infused rats in the final 60 minutes of the clamp(165±18 pM versus 298±26 pM, respectively; P<0.002). The hyperglycemicclamp protocol also enabled measurement of insulin sensitivity. Glucoseinfusion rate during the clamp was increased by 111±7% inexendin-4-treated versus control rats (P<0.001). In other words,exendin-4 exhibited a glucagonostatic effect in ZDF rats duringhyperglycemic clamp studies, an effect that will be of therapeuticbenefit in diabetic humans.

EXAMPLE 5 Metabolic Effects of Exendin-4 on Glucagon Secretion in Peoplewith Type 2 Diabetes

In this Example, the safety, tolerability, and efficacy of syntheticexendin-4 was evaluated in 8 male non-insulin using patients with type 2diabetes who had discontinued other antidiabetic therapy for a minimumof 7 days. Each patient received subcutaneous (SC) injections of placebo(PBO) and 0.1, 0.2, and 0.3 μg/kg exendin-4 48 hours apart in asingle-blind, dose-rising, placebo controlled crossover design. Fivepatients also received a 0.4 μg/kg dose. Plasma glucose, insulin andglucagon concentrations were assessed fasting and in response to a 7Kcal/kg Sustacal® challenge administered at the time of exendin-4/PBOinjection. Gastric emptying was evaluated by measuring serumacetaminophen concentrations following a 20 mg/kg oral dose of liquidacetaminophen administered with the Sustacal®. No safety issues wereidentified based upon reported adverse events, EKG and safety labmonitoring. Doses of 0.3 and 0.4 μg/kg elicited a dose-dependentincrease in nausea; vomiting occurred at the highest dose.

Plasma glucose concentrations were reduced in all doses of exendin-4compared to PBO although insulin concentrations were not significantlydifferent. The 8 hour mean±SE changes in plasma glucose AUC frombaseline were +391±187, −263±108, −247±64, −336±139, and −328±70mg*hr/dL for the PBO, 0.1, 0.2, 0.3, and 0.4 μg/kg doses respectively.The 3 hr changes in plasma glucagon were +128.0±19.2, −5.6±10.5,−29.4±18.6, −40.5±24.5, and +6.9±38.6 pg*hr/mL respectively. The gastricemptying rate was slowed in all doses and the mean total absorbedacetaminophen over 6 hours was reduced by 51%, 50%, 57% and 79% comparedto PBO for 0.1, 0.2, 0.3, and 0.4 μg/kg doses respectively. In summary,SC injection of exendin-4 to patients identified no safety issues, wastolerated at doses ≦0.3 μg/kg, reduced plasma glucose and glucagon andslowed the rate of gastric emptying. These observations support the useof exendin for the treatment of conditions that would benefit fromreduced glucagon levels and/or suppression of glucagon, including butnot limited to type 1 and type 2 diabetes.

EXAMPLE 6 PEG Modified Exendin 4

In the case of exendin 4, a 39 amino acid peptide with a molecularweight of 4187, modifications that increase its size and/or increase itsanionic nature will decrease its ability to be filtered by the kidney.Because clearance of exendin 4 is largely by the kidney this willeffectively increase its half life. Other properties of PEGylation(increased plasma half-life due to evasion of such renal and/or cellularclearance mechanisms that may exist; reduced immunogenicity andantigenicity; increased solubility; resistance to proteolysis; reducedtoxicity (avoid dose spike); improved thermal and mechanical stability;improved permeability of the mucus or epithelial layer; and selectivecontrol over a specific biological function) are also of potentialbenefit for exendin 4 and exendin agonists.

In particular, because we have observed multiple pharmacologies (likelyrepresenting multiple receptor subtypes), different spectra ofbiological activities of exendin 4 may be selected by putting a PEGgroup at appropriate positions. Loss or alteration of bioactivity hasbeen reported for PEGylated proteins which may be due to the presence ofthe PEG chains themselves, the particular site occupied by the PEGchain, or the coupling conditions having an adverse effect on theprotein.

Primary considerations for PEG modification in terms of filtration atthe kidney of exendin and exendin agonists are size and charge.Unmodified, exendin 4 has a molecular weight of approximately 4.2 kD andis anionic in nature with an overall net charge of approximately −2 atphysiological pH. One, two or three PEG constituents may be covalentlylinked to exendin 4 or an analog of exendin 4, for example, with one PEGconstituent being preferred. The size of the PEG can vary from amolecular weight of 500 to 20,000, preferably between 5,000 and 12,000.

Many of the methods for covalent attachment of PEG take advantage of theepsilon-amino group on lysine. Exendin 4 has two lysines that can bemodified by attachment of PEG. An alanine scan of AC3177 (Leu¹⁴,Phe²⁵1-28 exendin-4), a shortened analog of exendin 4, revealedpositions that are sensitive to substitution by alanine. The two lysinesat positions 12 and 27 were moderately affected by this substitutionsuggesting that loss of the lysine specific R group side chain(methylene chain plus epsilon-amino group) is tolerated. With regard tothe full-length peptide, exendin 4, the two lysine positions areappropriate for PEG attachment (see compounds 1 and 2). In addition,depending on the chemistry used to conjugate the PEG, the epsilon-aminogroups at these positions may be masked thereby increasing the anionicnature of the peptide. (201) HGEGTFTSDLSK (PEG)QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (202) HGEGTFTSDLSKQMEEEAVRLFIEWLK (PEG)NGGPSSGAPPPS-NH₂

Based on the results of the alanine scan, other likely positions thatmay be modified by insertion of a Lys-PEG or equivalent, for example,are: (203) HK (PEG) EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (204)HGEGK (PEG) FTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (205) HGEGTFTK (PEG)DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (206) HGEGTFTSDK (PEG)SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (207) HGEGTFTSDLK (PEG)KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (208) HGEGTFTSDLSKK (PEG)MEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (209)* HGEGTFTSDLSKQMEK (PEG)EAVRLFIEWLKNGGPSSGAPPPS-NH₂ (210)* HGEGTFTSDLSKQMEEK (PEG)AVRLFIEWLKNGGPSSGAPPPS-NH₂ (211) HGEGTFTSDLSKQMEEEAK (PEG)RLFIEWLKNGGPSSGAPPPS-NH₂ (212) HGEGTFTSDLSKQMEEEAVRK (PEG)FIEWLKNGGPSSGAPPPS-NH₂ (213)* HGEGTFTSDLSKQMEEEAVRLFIK (PEG)WLKNGGPSSGAPPPS-NH₂ (214) HGEGTFTSDLSKQMEEEAVRLFIEK (PEG)LKNGGPSSGAPPPS-NH₂ (215) HGEGTFTSDLSKQMEEEAVRLFIEWLKK (PEG)GGPSSGAPPPS-NH₂

The three positions* above normally containing a glutamic acid that wereindicated for modification with K(PEG) can also be modified byconjugation to the glutamic side chain carboxyl group, E(PEG).

Another analog in which the Lys-PEG can be added is at the supposedGlyGly turn: (216) HGEGTFTSDLSKQMEEEAVRLFIEWLKN K (PEG) GPSSGAPPPS-NH₂(217) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGK (PEG) PSSGAPPPS-NH₂

Positions 29-39 of exendin-4 may not be critical for the glucoselowering activity as evidenced by AC3177 having nearly equipotentactivity to exendin 4, and any of them, alone or in combination, can besubstituted for K(PEG) or an equivalent.

EXAMPLE 7 Exendin-4 is a Circulating, Meal-Related Peptide in the GilaMonster

This experiment investigated whether exendin-4 has a metabolic role inthe Gila monster lizard itself. To investigate whether exendin-4appeared in the blood of the Gila monster in response to feeding, bloodwas sampled from one animal fasted for 7 weeks, before and 30 min afteringestion of a small rat. Plasma was assayed for full-length exendin-4using an immunoradiometric assay with monoclonal antibody pairs directedto epitopes at N- and C-termini of exendin-4. Fasting plasma exendin-4concentration was 76 pg/mL, near the lower limit of quantitation. Aftereating, this value rose 300-fold to 23,120 pg/mL.

In a second experiment, serial samples were taken from two animalsfasted five weeks before and after ingestion of one or two small rats(47-49 g). Plasma exendin-4 concentration rose 23- to 36-fold (to 4860,8340 pg/mL) immediately after eating, consistent with a direct passageof exendin-4 from the salivary gland to blood. After eating a second rat(t=30 min), plasma exendin-4 concentration in one Gila rose further to27,209 pg/mL. Plasma exendin-4 concentration decayed with a t % of 5.00and 5.33 hours, respectively. In conclusion, exendin-4, known tooriginate from the salivary gland of the Gila monster, appears in highconcentration in the blood immediately after eating. This may representa meal-related signal to inhibit further eating and promote nutrientstorage.

EXAMPLE 8 Exendin-4 Decreases Glucagon Secretion During HyperglycemicClamps in Diabetic Fatty Zucker Rats

Absolute or relative hyperglucagonemia is often a feature of type 1 andtype 2 diabetes mellitus, and the suppression of excessive glucagonsecretion is a potential benefit of therapy using glucagonostaticagents. In this Example, the effect of exendin-4 on glucagon secretionin male anaesthetized Diabetic Fatty Zucker (ZDF) rats was examined.Using an hyperinsulinemic hyperglycemic clamp protocol, factors tendingto influence glucagon secretion were held constant. Plasma glucose wasclamped at ˜34 mM 60 min before beginning intravenous infusions ofsaline (n=7) or exendin-4 (0.21 μg+2.1 μg/mL/h; n=7). Plasma glucagonconcentration measured before these infusions were similar in bothgroups (306±30 pM versus 252±32 pM, respectively; n.s.).

Mean plasma glucagon concentration in exendin-4 infused rats was nearlyhalf of that in saline-infused rats in the final 60 minutes of the clamp(165±18 pM versus 298±26 pM, respectively; P<0.002). The hyperglycemicclamp protocol also enabled measurement of insulin sensitivity. Glucoseinfusion rate during the clamp was increased by 111±7% inexendin-4-treated versus control rats (P<0.001). In other words,exendin-4 exhibited a glucagonostatic effect in ZDF rats duringhyperglycemic clamp studies, an effect that will be of therapeuticbenefit in diabetic humans.

EXAMPLE 9 Pharmacokinetics of Exendin-4 in the Rat FollowingIntravenous, Subcutaneous and Intraperitoneal Administration

This Example describes work to define the plasma pharmacokinetics ofexendin-4 in rats (˜350 g body weight each) following 2.1, 21, 210μg/rat i.v. bolus, s.c. and i.p. administration and 2.1, 21, 210μg/hr/rat i.v. infusion (3 hr). Serial samples of plasma (˜120 μL) wereassayed using a validated immunoradiometric assay (IRMA). Thissandwich-type assay uses mouse-based monoclonal antibodies that reactwith exendin-4 but do not react with GLP-1 or tested metabolites ofexendin-4 or GLP-1. The lower limit of quantitation was 15 pM (63pg/mL). The estimated t_(1/2) for exendin-4 was 18-41 min for i.v.bolus, 28-49 for i.v. continuous, 90-216 min for s.c. and 125-174 minfor i.p. injection. Bioavailability was 65-76% for s.c. and i.p.injection. Clearance determined from the i.v. infusion was 4-8 mL/min.Both C_(max) and AUC values within each route of administration wereproportional to dose. Volume of distribution was 457-867 mL. Clearanceand bioavailability were not dose dependent. C_(max) (or steady-stateplasma concentration; C_(ss)) is shown in the table below Cmax or Css(nM) Route Dose IV bolus IV infusion Subcutaneous IP  2.1 μg   2.9 ± 0.41.1 ± 0.1 0.56 ± 0.12 0.26 ± 0.04  21 μg 70 ± 3  19 ± 1.9 4.1 ± 1.5 3.9± 1   210 μg 645 ± 12 262 ± 60  28 ± 4  35 ± 6 

EXAMPLE 10 Comparison of the Insulinotropic Actions of Exendin-4 andGlucagon-Like Peptide-1 (GLP-1) During an Intravenous Glucose Challengein Rats

This experiment compares the insulinotropic actions of syntheticexendin-4 and GLP-1 in vivo following an intravenous (i.v.) glucosechallenge in rats. Sprague-Dawley rats (˜400 g) were anesthetized withhalothane and cannulated via the femoral artery and saphenous vein.Following a 90-min recovery period, saline or peptide (30 pmol/kg/mineach) was administered i.v. (1 ml/h for 2 hours; n=4-5 for each group).Thirty min after infusion commenced, D-glucose (5.7 mmol/kg, 0.8 ml) wasinjected i.v. In saline-treated, exendin-4-treated and GLP-1-treatedrats, plasma glucose concentrations were similar before injection(9.3±0.3, 9.7±0.3, 10.3±0.4 mM), increased by similar amounts afterglucose injection (21.7, 21.3, 23.7 mM), and resulted in a similar60-min glucose AUC (987±39, 907±30, 1096±68 mM·min, respectively). Thatis, the glycemic stimulus was similar in each treatment group. Plasmainsulin concentration in saline-treated rats increased 3.3-fold with theglucose challenge (230±53 to a peak of 765±188 pM). With exendin-4infusion, the increase in plasma insulin concentration was 6.8-fold(363±60 to 2486±365 pM). With GLP-1 the increase in plasma insulinconcentration was 2.9-fold (391±27 to 1145±169 pM), which was similar tothat obtained in saline-treated rats. The 60-min insulin AUC insaline-treated rats was 24±6 nM·min, was increased 2.8-fold inexendin-treated rats (67±8 nM·min; P<0.003 versus saline; P<0.02 versusGLP-1) and by 20% in GLP-1-treated rats (n.s. versus saline).Amplification of glucose-stimulated insulin release by exendin-4 wasalso tested at infusion rates of 3 and 300 pmol/kg/min and shown to bedose-dependent. Thus, exendin-4 is more potent and/or effective thanGLP-1 in amplifying glucose-stimulated insulin release in intact rats.

EXAMPLE 11 Development and Validation of an Immunoradiometric Assay(IRMA) for the Quantitation of Exendin-4 in Plasma and its Applicationto Preclinical Toxicity and Phase I Clinical Evaluations

A sensitive and specific sandwich-type immunoradiometric (IRMA) assaywas developed for quantitation of plasma exendin-4 concentration usingsynthetic exendin-4 as the immunogen. One mouse-derived monoclonalantibody recognizes a C-terminal epitope on exendin-4 (capture antibody)but does not cross-react with GLP-1. The second antibody (detectorantibody labeled with ¹²⁵I) recognizes an N-terminal epitope onexendin-4 and GLP-1, and requires a terminal histidine for binding.Thus, the assay as a whole does not detect GLP-1(7-36)NH₂,GLP-1(7-36)COOH or exendin(3-39). Assay validation in rat, monkey, dog,rabbit and human plasmas showed inter- and intra-assay coefficients ofvariation <20% and <10%, respectively, accuracy of +15% with target low,mid and high controls, and lower and upper limits of quantitation of62.8 and 2512 pg/mL, respectively. Plasma samples from 28-daysubcutaneous toxicity evaluations of exendin-4 in rats and monkeys and aPhase I clinical study in normal subjects were evaluated using the IRMA.The C_(max) values in the animals studies are shown in the table below.Human samples from subcutaneous administration of 0.05, 0.1, 0.2 and 0.3μg/kg yielded C_(max) values of 90, 224, 370 and 587 pg/mL. Cmax (pg/mL)Dose (μg/kg) 10 100 1000 Rat 7,000 127,000 1,180,000 Monkey 20,000170,000 1,890,000

EXAMPLE 12 Comparison of GLP-1 Receptor Binding/Activating andGlucose-Lowering Effects of GLP-1 and Exendin-4

Exendin-4 was synthesized by solid phase peptide synthesis techniquesand compared to synthetic GLP-1 in terms of in vitro binding to, andactivation of, GLP-1 receptors, and in vivo in terms of lowering plasmaglucose in diabetic db/db mice. In a plasma membrane preparation of arat insulinoma cell line (RINm5f) that expresses the GLP-1 receptor, thepeptides were assayed for their ability to bind and displaceradiolabeled GLP-1 and for their ability to stimulate the production ofcAMP. The relative order of binding potency was found to beGLP-1>exendin-4. The relative order of cyclase activation wasGLP-1=exendin-4. Affinities, as shown in the table below, differ over a4- to 5-fold range. In contrast, in vivo glucose lowering potencydiffered over a 3430-fold range. Exendin-4 was 3430-fold more potentthan GLP-1. The in vivo potency of exendin-4 does not match potency atthe GLP-1 receptor, and is likely the culmination of an aggregate ofproperties. Binding Cyclase Glucose-lowering IC50 (nM EC50 (nM) ED50(μg) GLP-1 0.15 0.28 20.6 Exendin-4 0.66 0.30 0.006

EXAMPLE 13 Comparison of Glycemic Indices and Insulin Sensitivity inPair-Fed and Exendin-4-Treated Diabetic Fatty Zucker Rats

This Example tests whether the beneficial effects of exendin-4 in ZDFrats were secondary to changes in food intake. It compares effectsobtained with exendin-4 to effects observed in saline-treated matchedanimals who consumed the same amount of food as was eaten by ZDF ratsinjected subcutaneously twice daily with 10 μg exendin-4. Plasma glucoseand HbA1c were measured weekly for 6 weeks. One day after the lasttreatment, animals were anesthetized with halothane and subjected to anhyperinsulinemic (50 mU/kg/min) euglycemic clamp. Changes in HbA1c over6 weeks differed between treatment groups (P<0.001 ANOVA), increasing inad lib fed (n=5) and pair fed (n=5) rats, but decreasing inexendin-4-treated rats (n=5). Similarly, changes in plasma glucosediffered between treatment groups (P<0.002 ANOVA), increasing in ad libfed and pair fed ZDF rats, and decreasing in ZDF rats treated withexendin-4. In the final hour of a 3-hour clamp protocol, glucoseinfusion rate in exendin-4-treated rats tended to be higher than in pairfed (+105%) and ad lib fed (+20%) controls, respectively (10.14±1.43n=5, 8.46±0.87 n=4, 4.93±2.02 mg/kg/min n=3; n.s. P=0.09 ANOVA). Anotherindex of insulin sensitivity, plasma lactate concentration, differedsignificantly between treatment groups (P<0.02 ANOVA) and was lowest inexendin-4-treated rats. Thus, exendin-4 treatment is associated withimprovement in glycemic indices and in insulin sensitivity that ispartly, but not fully, matched in controls fed the same amount of food,indicating that improvements in metabolic control with exendin-4 in ZDFrats are at least partly due to mechanisms beyond caloric restriction.

EXAMPLE 14 Clinical Studies and the Stimulation of Endogenous InsulinSecretion by Subcutaneous Synthetic Exendin-4 in Healthy OvernightFasted Volunteers

In a double blind, placebo-controlled single ascending dose clinicaltrial to explore safety and tolerability and pharmacokinetics ofsynthetic exendin-4, exendin-4 formulated for subcutaneous injection wasevaluated in healthy male volunteers while assessing effects upon plasmaglucose and insulin concentrations. Five single subcutaneous doses ofexendin-4 (0.01, 0.05, 0.1, 0.2 or 0.3 μg/kg) were studied in 40 healthymale volunteers in the fasting state. Maximum plasma exendin-4concentrations were achieved between 1 and 2 hours post-dose with littledifference among the doses examined. Examination of the data indicated adose dependent increase for C_(max). There were no serious adverseevents reported in this study and in the healthy male volunteers thatparticipated in this study, exendin-4 was well tolerated at subcutaneousdoses up to and including 0.1 μg/kg. A decrease in plasma glucoseconcentration was also observed at this dose. At doses of 0.2 μg/kg andhigher, the most commonly observed adverse events were headache, nausea,vomiting, dizziness, and postural hypotension. There was a transientfall in plasma glucose concentration following administration of dosesof 0.05 μg/kg and above.

Forty healthy, lean (mean BMI (±SE) 22.7±1.2) subjects aged 18-40 yearswere randomly assigned to 5 groups. Within each group of 8 subjects, 6were assigned to exendin-4 and 2 to placebo (PBO). Exendin-4 (0.01,0.05, 0.1, 0.2 or 0.3 μg/kg) or placebo was administered following anovernight fast and plasma exendin-4, glucose and insulin concentrationsmonitored along with safety and tolerability. No safety issues wereobserved. Doses ≦0.1 μg/kg were tolerated as well as PBO whereas 0.2 and0.3 μg/kg elicited a dose-dependent increase in nausea and vomiting.Peak plasma exendin-4 concentrations rose dose-dependently and following0.1 μg/kg, exendin-4 immunoreactivity persisted for 360 min. Plasmaglucose decreased following all doses, except 0.01 μg/kg, reached anadir by 30 min and returned back to baseline within 180 min. Subjectsreceiving 0.3 μg/kg received a caloric beverage 30 minutes after dosing,precluding comparison of their data. Mean change in plasma glucose(0-180 min): 0.03±0.07, −0.07±0.08, −0.38±0.14, −0.85±0.13 and−0.83±0.23 mmol/L for PBO, 0.01, 0.05, 0.1, and 0.2 μg/kg respectively;P≦0.02 versus PBO. The lowest plasma glucose recorded was 3.4 mmol/L.Corresponding mean changes in plasma insulin (0-120 min) were 0.43±0.59,2.37±0.58, 2.28±0.66, 4.91±1.23, and 14.00±3.34 μU/mL; P≦0.01 versus PBOfor the 0.1 and 0.2 μg/kg groups. Thus, in healthy, overnight fastedvolunteers, subcutaneous injection of exendin-4 (1) presented no safetyissues, (2) was well-tolerated at doses ≦0.1 μg/kg, (3) led to exendin-4immunoreactivity in plasma for up to 6 hrs, (4) increased plasma insulinand lowered plasma glucose in a dose-dependent manner without inducinghypoglycemia.

EXAMPLE 15 Effectiveness of Alternate Delivery of Exendin-4 in Rodents

This Example tested the delivery of exendin-4 by means alternative toinjection, and examined its ability to traverse mucosal surfaces insufficient quantities to exert biological effect. Changes inconcentration of plasma glucose and of intact synthetic exendin-4(measured by a 2-site immunoradiometric assay) were observed in db/dbmice administered a saline solution containing differing doses ofsynthetic exendin-4 via the trachea, via an aerosol mist (pulmonary),via gavage (oral), and under the tongue (sublingual). The same routes ofadministration, as well as intraduodenally and nasally, were tested inrats, and bioavailability was calculated, for example, for sublingualand intra-tracheal routes. Exendin-4 administered via each of the aboveroutes in mice resulted in significant glucose-lowering activity 1 to 4hours after administration (db/db mice intra-tracheal P<0.02; ob/ob miceintra-tracheal P<0.0002; db/db mice aerosol P<0.0001; gavage P<0.002;sublingual P<0.02). Dose-dependent increases in plasma exendin-4concentration were up to 777±365 pg/mL (db/db mice intra-tracheal);170±67 pg/mL (db/db mice aerosol); 4520±1846 pg/mL (db/db micesublingual). Similarly, in rats, exendin-4 concentrations were observedup to 68,682±38,661 pg/mL (intra-tracheal); 1900 pg/mL (pulmonary); 6757pg/mL (nasal); 3,862±2,844 pg/mL (sublingual); but no apparentabsorption or biological activity when delivered intraduodenally.Bioavailability of exendin-4 in saline was ˜7.3% at lower doses whendelivered via the trachea, where 61-74% of Cmax was observed within 5min. Kinetics thereafter were similar to those observed aftersubcutaneous administration. Bioavailability of exendin-4 in salinedelivered under the tongue was 3.1-9.6% at lower doses. These studiessupport the delivery of exendin-4 and peptide agonist analogs thereof inbiologically effective quantities via convenient non-injectable routes.

EXAMPLES 16 to 20

Reagents Used

GLP-1[7-36]NH₂ (GLP-1) was purchased from Bachem (Torrance, Calif.). Allother peptides were prepared using synthesis methods such as thosedescribed therein. All chemicals were of the highest commercial grade.The cAMP SPA immunoassay was purchased from Amersham. The radioligandswere purchased from New England Nuclear (Boston, Mass.). RINm5f cells(American Type Tissue Collection, Rockville, Md.) were grown in DME/F12medium containing 10% fetal bovine serum and 2 mM L-glutamine. Cellswere grown at 37° C. and 5% CO₂/95% humidified air and medium wasreplaced every 2 to 3 days. Cells were grown to confluence thenharvested and homogenized using on a Polytron homogenizer. Cellhomogenates were stored frozen at −70° C. until used.

EXAMPLE 16 GLP-1 Receptor Binding Studies

Receptor binding was assessed by measuring displacement of [¹²⁵I]GLP-1or [¹²⁵I]exendin(9-39) from RINm5f membranes. Assay buffer contained 5μg/ml bestatin, 1 μg/ml phosphoramidon, 1 mg/ml bovine serum albumin(fraction V), 1 mg/ml bacitracin, and 1 mM MgCl₂ in 20 mM HEPES, pH 7.4.To measure binding, 30 μg membrane protein (Bradford protein assay) wasresuspended in 200 μl assay buffer and incubated with 60 pM [¹²⁵I]GLP-1or [¹²⁵I]exendin(9-39) and unlabeled peptides for 120 minutes at 23° C.in 96 well plates (Nagle Nunc, Rochester, N.Y.). Incubations wereterminated by rapid filtration with cold phosphate buffered saline, pH7.4, through polyethyleneimine-treated GF/B glass fiber filters (WallacInc., Gaithersburg, Md.) using a tomtec Mach II plate harvester (WallacInc., Gaithersburg, Md.). Filters were dried, combined with scintillant,and radioactivity determined in a Betaplate liquid scintillant counter(Wallac Inc.).

Peptide samples were run in the assay as duplicate points at 6 dilutionsover a concentration range of 10⁻⁶M to 10⁻¹²M to generate responsecurves. The biological activity of a sample is expressed as an IC₅₀value, calculated from the raw data using an iterative curve-fittingprogram using a 4-parameter logistic equation (Prizm, GraphPADSoftware).

EXAMPLE 17 Cyclase Activation Study

Assay buffer contained 10 μM GTP, 0.75 mM ATP, 2.5 mM MgCl₂, 0.5 mMphosphocreatine, 12.5 U/ml creatine kinase, 0.4 mg/ml aprotinin, 1 μMIBMX in 50 mM HEPES, pH 7.4. Membranes and peptides were combined in 100ml of assay buffer in 96 well filter-bottom plates (Millipore Corp.,Bedford, Mass.). After 20 minutes incubation at 37° C., the assay wasterminated by transfer of supernatant by filtration into a fresh 96 wellplate using a Millipore vacuum manifold. Supernatant cAMP contents werequantitated by SPA immunoassay. Peptide samples were run in the assay astriplicate points at 7 dilutions over a concentration range of 10⁻⁶M to10⁻¹²M to generate response curves. The biological activity of aparticular sample was expressed as an EC₅₀ value, calculated asdescribed above.

EXAMPLE 18 Determination of Blood Glucose Levels in db/db Mice

C57BLKS/J-m-db mice at least 3 months of age were utilized for thestudy. The mice were obtained from The Jackson Laboratory and allowed toacclimate for at least one week before use. Mice were housed in groupsof ten at 22° C.±1° C. with a 12:12 light:dark cycle, with lights on at6 a.m. All animals were deprived of food for 2 hours before takingbaseline blood samples. Approximately 70 μl of blood was drawn from eachmouse via eye puncture, after a light anesthesia with metophane. Aftercollecting baseline blood samples, to measure plasma glucoseconcentrations, all animals receive subcutaneous injections of eithervehicle (10.9% NaCl), exendin-4 or test compound (1 μg) in vehicle.Blood samples were drawn again, using the same procedure, after exactlyone hour from the injections, and plasma glucose concentrations weremeasured. For each animal, the % change in plasma value, from baselinevalue, was calculated.

EXAMPLE 19 Dose Response Determination of Blood Glucose Levels in db/dbMice

C57BLKS/J-m-db/db mice, at least 3 months of age were utilized for thestudy. The mice were obtained from The Jackson Laboratory and allowed toacclimate for at least one week before use. Mice were housed in groupsof ten at 22° C.±1° C. with a 12:12 light:dark cycle, with lights on at6 a.m. All animals were deprived of food for 2 hours before takingbaseline blood samples. Approximately 70 μl of blood was drawn from eachmouse via eye puncture, after a light anesthesia with metophane. Aftercollecting baseline blood samples, to measure plasma glucoseconcentrations, all animals receive subcutaneous injections of eithervehicle, exendin-4 or test compound in concentrations indicated. Bloodsamples were drawn again, using the same procedure, after exactly onehour from the injections, and plasma glucose concentrations weremeasured. For each animal, the % change in plasma value, from baselinevalue, was calculated and a dose dependent relationship was evaluatedusing Graphpad Prizm™ software.

EXAMPLE 20 Gastric Emptying

The following study was and may be carried out to examine the effects ofexendin-4 and/or an exendin agonist compound on gastric emptying inrats. This experiment followed a modification of the method ofScarpignato, et al., Arch. Int. Pharmacodyn. Ther. 246:286-94, 1980.Male Harlan Sprague Dawley (HSD) rats were used. All animals were housedat 22.7±0.8 C in a 12:12 hour light:dark cycle (experiments beingperformed during the light cycle) and were fed and watered ad libitum(Diet LM-485, Teklad, Madison, Wis.). Exendin-4 was synthesizedaccording to standard peptide synthesis methods. The preparation ofexendin-4 is described in Example 2. The determination of gastricemptying by the method described below was performed after a fast of ˜20hours to ensure that the stomach contained no chyme that would interferewith spectrophotometric absorbance measurements.

Conscious rats received by gavage, 1.5 ml of an acaloric gel containing1.5% methyl cellulose (M-0262, Sigma Chemical Co, St Louis, Mo.) and0.05% phenol red indicator. Twenty minutes after gavage, rats wereanesthetized using 5% halothane, the stomach exposed and clamped at thepyloric and lower esophageal sphincters using artery forceps, removedand opened into an alkaline solution which was made up to a fixedvolume. Stomach content was derived from the intensity of the phenol redin the alkaline solution, measured by absorbance at a wavelength of 560nm. In separate experiments on 7 rats, the stomach and small intestinewere both excised and opened into an alkaline solution. The quantity ofphenol red that could be recovered from the upper gastrointestinal tractwithin 20 minutes of gavage was 89±4%; dye which appeared to bindirrecoverably to the gut luminal surface may have accounted for thebalance. To account for a maximal dye recovery of less than 100%,percent of stomach contents remaining after 20 min were expressed as afraction of the gastric contents recovered from control rats sacrificedimmediately after gavage in the same experiment. Percent gastriccontents remaining=(absorbance at 20 min)/(absorbance at 0 mm)×100.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitations,which is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other embodimentsare within the following claims.

1. A pharmaceutical formulation which is a liquid dosage form suitablefor multi-use administration comprising about 0.005% (w/v) to about 0.4%(w/v) of an exendin, an exendin analog or a combination thereof, abuffer, an iso-osmolality modifier, and about 0.005% to about 1.0% (w/v)of a preservative selected from the group consisting of m-cresol,phenol, benzyl alcohol, methyl-, ethyl-, propyl- and butyl-paraben andany combination thereof, wherein said formulation has a pH of betweenabout 3.0 and about 7.0 and said exendin is not exendin-4.
 2. Thepharmaceutical formulation of claim 1, where said exendin is exendin-3.3. The pharmaceutical formulation of claim 1, where said exendin analogcomprises SEQ ID NO.
 41. 4. The pharmaceutical formulation of claim 1,where said exendin analog comprises SEQ ID NO.
 42. 5. The pharmaceuticalformulation of claim 1, where said exendin analog comprises SEQ ID NO.43.
 6. The pharmaceutical formulation of claim 1, where said exendinanalog comprises SEQ ID NO.
 44. 7. The pharmaceutical formulation ofclaim 1, where said exendin analog comprises SEQ ID NO.
 45. 8. Thepharmaceutical formulation of claim 1, where said exendin analogcomprises SEQ ID NO.
 46. 9. The pharmaceutical formulation of claim 1,where said exendin analog comprises SEQ ID NO.
 47. 10. Thepharmaceutical formulation of claim 1, where said exendin analogcomprises SEQ ID NO.
 48. 11. The pharmaceutical formulation of claim 1,where said exendin analog is selected from at least one of the groupconsisting of SEQ ID NOs 6-189.
 12. The pharmaceutical formulation ofclaim 1, wherein said exendin analog is selected from at least one ofthe group consisting of SEQ ID NOs 6, 7, 8, 9, 40 and
 41. 13. Theformulation of claim 1, said formulation having a pH of between about4.0 and about 6.0.
 14. The formulation of claim 1, wherein said pH isbetween about 4.0 and about 5.0.
 15. The formulation of claim 1, whereinsaid exendin or exendin analog is present at a concentration of betweenabout 0.005% (w/v) and about 0.05% (w/v).
 16. The formulation of claim1, wherein said buffer is selected from the group consisting of anacetate buffer, a glutamate buffer, a citrate buffer, a phosphatebuffer, and any combination thereof.
 17. The formulation of claim 1,wherein said buffer is at a concentration between about 0.02% (w/v) andabout 0.5% (w/v).
 18. The formulation of claim 1, wherein saidiso-osmolality modifier is a carbohydrate, a polyhydric alcohol, or acombination thereof, and said iso-osmolality modifier is at aconcentration between about 1% (w/v) and 10% (w/v).
 19. The formulationof claim 18, wherein said polyhydric alcohol is selected from the groupconsisting of sorbitol, mannitol, inositol, glycerol, xylitol,polyethylene glycols, and any combination thereof.
 20. The formulationof claim 18, wherein said carbohydrate is selected from the groupconsisting of galactose, arabinose, lactose, and any combinationthereof.
 21. The formulation of claim 1, wherein said iso-osmolalitymodifier is mannitol, sorbitol, or a combination thereof.
 22. Theformulation of claim 1, wherein said preservative is m-cresol, phenol ora combination thereof.
 23. The formulation of claim 1, wherein saidformulation is suitable for administration via injection to achieve adose of from about 0.1 μg/kg to about 0.5 μg/kg of said exendin orexendin analog.
 24. The formulation of claim 1, wherein said formulationis suitable for administration via injection to achieve a dose of fromabout 0.005 μg/kg to about 0.2 μg/kg of said exendin or exendin analog.25. The formulation of claim 1, wherein said formulation is suitable foradministration via injection to achieve a dose of from about 1 μg/day toabout 1 mg/day of said exendin or exendin analog.
 26. A pharmaceuticalformulation which is a liquid dosage form suitable for multi-useadministration comprising an exendin, an exendin analog or a combinationthereof, a buffer, an iso-osmolality modifier, and about 0.005% (w/v) toabout 1.0% (w/v) of a preservative selected from the group consisting ofm-cresol, phenol, benzyl alcohol, methyl-, ethyl-, propyl- andbutyl-paraben and any combination thereof, wherein said formulation hasa pH of between about 3.0 and about 6.0 and said exendin is notexendin-4.
 27. The formulation of claim 26, wherein said formulation issuitable for a route of administration selected from the groupconsisting of: oral administration to achieve a dose of from about 500μg/day to about 12,000 μg/day of said exendin or exendin analog in asingle or divided dose; pulmonary administration to achieve a dose fromabout 100 μg/day to about 12,000 μg/day of said exendin or exendinanalog in a single or divided dose; sublingual administration to achievea dose from about 10 μg/day to about 8,000 μg/day of said exendin orexendin analog in a single or divided dose; nasal administration toachieve a dose from about 10 μg/day to about 12,000 μg/day of saidexendin or exendin analog in a single or divided dose; and buccaladministration to achieve a dose from about 100 μg/day to about 12,000μg/day of said exendin or exendin analog in a single or divided dose.28. The formulation of claim 26, said formulation having a pH of betweenabout 4.0 and about 6.0.
 29. The formulation of claim 26, wherein saidpH is between about 4.0 and about 5.0.
 30. The formulation of claim 26,wherein said buffer is selected from the group consisting of an acetatebuffer, a glutamate buffer, a citrate buffer, a phosphate buffer, andany combination thereof.
 31. The formulation of claim 26, wherein saidbuffer is at a concentration between about 0.02% (w/v) and about 0.5%(w/v).
 32. The formulation of claim 26, wherein said iso-osmolalitymodifier is a carbohydrate, a polyhydric alcohol, or a combinationthereof, and said iso-osmolality modifier is at a concentration betweenabout 1% (w/v) and 10% (w/v).
 33. The formulation of claim 26, whereinsaid polyhydric alcohol is selected from the group consisting ofsorbitol, mannitol, inositol, glycerol, xylitol, polyethylene glycols,and any combination thereof.
 34. The formulation of claim 26, whereinsaid carbohydrate is selected from the group consisting of galactose,arabinose, lactose, and any combination thereof.
 35. The formulation ofclaim 26, wherein said iso-osmolality modifier is mannitol, sorbitol, ora combination thereof.
 36. The formulation of claim 26, wherein saidpreservative is m-cresol, phenol or a combination thereof.